Methods of generating, repairing and/or maintaining connective tissue in vivo

ABSTRACT

This invention relates to a method for generating, repairing and/or maintaining connective tissue in a subject. In one embodiment, the invention relates to a method for generating, repairing and/or maintaining cartilage tissue in a subject. The present invention also relates to a method of treating and/or preventing a disease in a subject arising from degradation and inflammation of connective tissue.

FIELD OF THE INVENTION

This invention relates to a method for generating, repairing and/ormaintaining connective tissue in a subject. The present invention alsorelates to a method of treating and/or preventing a disease in a subjectarising from degradation and inflammation of connective tissue.

BACKGROUND OF THE INVENTION

Non-hematopoietic progenitor cells that reside in the body and give riseto multipotential cells when isolated are referred to as MesenchymalPrecursor Cells (MPCs). More specifically, purified MPCs are capable offorming very large numbers of multipotential cell colonies.

Simmons et al. (1994) describes enrichment of MPCs from freshlyharvested bone marrow cells by selecting for cells that express theSTRO-1 cell surface marker. As explained by the authors at pages272-273, it is known that bone marrow cells contain a proportion of MPCsthat are capable of giving rise to CFU-F. These CFU-F in turn arecapable of giving rise under appropriate conditions to a broad spectrumof fully differentiated connective tissue, including cartilage, bone,adipose tissue, fibrous tissue and myelosupportive stroma.

MPCs and CFU-F are typically present at a very low incidence in bonemarrow cells (typically between 0.05%-0.001%) and this rarity has been amajor limitation to their study in the past. An important findingdiscussed by Simmons et al. (1994) was the identification that theseMPCs could be enriched from freshly isolated bone marrow cells to someextent by selecting for STRO-1 positive cells. In particular, theselection of STRO-1 positive cells enabled isolation of MPCs (andresultant CFU-F) free of contaminating hemopoietic progenitors.

WO 01/04268 provided a further important advance in the enrichment ofMPCs by identifying a subpopulation within this fraction of STRO-1positive cells that contains MPCs. In particular, WO 01/04268 describesthe sorting of the STRO-1 positive cell population into three subsets:STRO-1^(dull), STRO-1^(intermediate) and STRO-1^(bright). Clonogenicassays for CFU-F in the different sorted subpopulations demonstratedthat the vast majority of the MPCs are contained within theSTRO-1^(bright) fraction.

WO 2004/085630 discloses for the first time that MPCs are present inperivascular tissue. One of the benefits of this finding is that itgreatly expands the range of source tissues from which MPCs can beisolated or enriched and there is no longer an effective restriction onthe source of MPCs to bone marrow. The tissues from which MPCs can beisolated according to the methods described in WO 2004/085630 includehuman bone marrow, dental pulp, adipose tissue, skin, spleen, pancreas,brain, kidney, liver and heart. The MPCs isolated from perivasculartissue are positive for the cell surface marker 3G5. They can thereforebe isolated by enriching for cells carrying the 3G5 marker, or byenriching for an early developmental surface marker present onperivascular cells such as CD146 (MUC18), VCAM-1, or by enriching forhigh level expression of the cell surface marker STRO-1.

The avascular connective tissues are generally located at anatomicalsites within the musculoskeletal system that require appreciablemovement. These freely movable joints are responsible for the majorityof articulations in mammals. In synovial joints the contact surfaces oftwo opposing bones are covered by hyaline cartilages which glideeffortlessly over each other because of the presence of a low frictionlubricant in synovial fluid produced by the cells lining the jointcapsule which overlays and connects the long bones. In the spinal columnarticulation is achieved by connection of the rigid vertebral bones bymeans of a flexible fibrocartilagenous ring (the annulus fibrosus) thatencapsulates a hydrated gelatinous mass (the nucleus pulposus),populated by chondrocyte like cells similar to those present in hyalinecartilage. Irrespective of the type and location of these avascularconnective tissue they all contain cells which synthesise anextracellular matrix which is rich in highly negatively chargedproteoglycans, which imbibe water molecules together with the fibrousprotein, type II collagen, which confers high tensile strength.

Avascular connective tissues such as hyaline cartilage, the inner twothirds of the meniscus and the intervertebral disc have limited repaircapabilities and when injured may respond by the production of afunctionally inferior fibrocartilagenous scar tissue. Through amultitude of factors, dominated by aging, genetics, hormonal status andphysical injury these avascular connectives often fail leading to thewidespread clinical problems of disc degeneration, back pain andosteoarthritis.

Current medical therapies normally used to treat the symptoms arisingfrom the failure of these connective tissues, for the most part, dolittle to redress the underlying pathology responsible for producing thesymptoms and in many instances may even exacerbate the problem by downregulating the capacity of the resident cells to synthesis thestructural components of the tissue extracellular matrix. Ideally,therapeutic treatments should be at least chondroprotective but evenprovide the conditions which enhance matrix biosynthesis and effectrepair and restoration of the injured connective tissues.

SUMMARY OF THE INVENTION

The present inventors have now made the surprising finding thatintra-articular administration of MPCs provides a chondroprotectiveeffect in joints with pre-existing osteoarthritis, and leads togeneration and growth of cartilage tissue in synovial joints and in thenucleus pulposus of the intervertebral discs. This finding indicatesthat MPCs or their progeny, or supernatant or soluble factors derivedfrom these MPCs, can be used to protect or repair damaged connectivetissues as well as generate new functional tissue at sites ofdegeneration or injury.

Accordingly, the present invention provides a method of treating and/orpreventing a disease in a subject arising from degradation and/orinflammation of connective tissue, the method comprising administeringto the subject MPCs and/or progeny cells thereof and/or soluble factorsderived therefrom.

In one embodiment of the invention, the connective tissue is rich inproteoglycans. The connective tissue may be cartilage, for example,hyaline cartilage. In another embodiment, the disease results in adefect in the cartilage.

In another embodiment, the method comprises administering to the subjectMPCs and/or progeny cells thereof and/or soluble factors derivedtherefrom, wherein the MPCs and/or progeny cells and/or soluble factorsare not directly administered into the defect.

For example, administration may me made into a joint space in order totreat or prevent defects in the cartilage on the articular surfaces ofbones that form that joint. Similarly, administration may be made intoan invertebral disc space in order to treat or prevent defects in thesurrounding discs. In another example, administration is madeintravenously at a site near the cartilage defect.

The MPCs and/or progeny cells and/or soluble factors may be administeredby intra-articular injection. The intra-articular injection may be madeinto any joint of the body which is near to a site of a cartilagedefect, or a potential cartilage defect. For example, theintra-articular injection may be made into a knee joint, hip joint,ankle joint, shoulder joint, elbow joint, wrist joint, hand or fingerjoint or a joint of the foot, or an invertebral disc joint.

In another embodiment of the invention, administration of the MPCsand/or progeny cells and/or soluble factors results in preservation orgeneration of cartilage that is rich in proteoglygans and type IIcollagen. An example of a cartilage that is rich in proteoglycans andtype II collagen is hyaline cartilage. Preferably the cartilagepreserved or generated by the method of the present invention is notfibrocartilage, which is rich in type I collagen, very low in type IIcollagen and contains less proteoglycan than hyaline cartilage.

Examples of diseases “arising from degradation and/or inflammation ofconnective tissue” include, but are not limited to, tendonitis, backpain, rotary cuff tendon degradation, Carpal tunnel syndrome,DeQuervain's syndrome, degenerative cervical and/or lumber discs,intersection syndrome, reflex sympathetic dystrophy syndrome (RSDS),stenosing tenosynovitis, epicondylitis, tenosynovitis, thoracic outletsyndrome, ulnar nerve entrapment, radial tunnel syndrome, repetitivestrain injury (RSI). Examples of diseases that are associated withdegradation and/or inflammation of hyaline cartilage include, but arenot limited to arthritis such as osteoarthritis, rheumatoid arthritis,psoriatic arthritis, and seronegative arthritis, arthritis associatedwith inflammatory bowel disease or ankylosing spondylitis and degenerateinvertebral disc disorders.

In another preferred embodiment, the method further comprisesadministering hyaluronic acid (HA). HA can be administered in the sameor different composition as the cells, supernatant and/or factor(s).

The present invention also provides a composition comprising MPCs and/orprogeny cells thereof and hyaluronic acid.

The results presented herein indicate for the first time that solublefactors released by the implanted cultured MPCs are supportive ofconnective tissue protection, generation and growth.

Accordingly, the present invention also provides a compositioncomprising;

i) supernatant, or one or more soluble factors, derived from mesenchymalprecursor cells (MPCs) and/or progeny cells thereof, and

ii) hyaluronic acid.

In a further aspect, the present invention provides for the use ofsupernatant, or one or more soluble factors, derived from mesenchymalprecursor cells (MPCs) and/or progeny cells thereof for treating and/orpreventing a disease in a subject arising from degradation and/orinflammation of connective tissue.

The present invention is applicable to a wide range of animals. Forexample, the subject may be a mammal such as a human, dog, cat, horse,cow, or sheep. In one embodiment the subject is a human.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

The invention is hereinafter described by way of the followingnon-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Means±SD of femoral and tibial cartilage morphology scores 12weeks post-meniscectomy for joints injected with Hyaluronan (HA) or HAplus different doses of Mesenchymal Precursor Cells (MPC).

FIG. 2. Means±SD of femoral and tibial osteophyte scores 12 weekspost-meniscectomy for joints injected with Hyaluronan (HA) or HA plusdifferent doses of Mesenchymal Precursor Cells (MPC).

FIG. 3. Ratios [HA/(MPC+HA)] of cartilage morphology joint scores foranimals injected with different doses of Mesenchymal Precursor Cells(MPC). When ratio=1 both treatments equally effective. Ratios>1 indicateMPC+HA superior to HA.

FIG. 4. Ratios [HA/(MPC+HA)] of osteophyte scores for animals injectedwith different doses of MPC+HA relative to HA alone. When ratio=1 bothtreatments equally effective. Ratios>1 indicate MPC+HA superior to HA.

FIG. 5. Means±SE of histomorphometrically determined regional thicknessscores for cartilages of joints injected with hyaluronan (HA) or 100million MPC+HA twelve weeks post meniscectomy. Combining all tibialcartilage regions HA+100 million MPC>HA (p<0.05).

FIG. 6. Ratios [HA/(MPC+HA)] of mean±SE total Mankin Modified jointhistopathology scores for animals injected with different doses ofMesenchymal Precursor Cells (MPC). When ratio=1 both treatments equallyeffective. Ratios>1 indicate MPC+HA superior to HA.

FIG. 7. Means±SD of femoral and tibial cartilage morphology scores forHA and HA+100 million MPC injected joints 12, 24 and 52 weeks postmeniscectomy.

FIG. 8. Means±SD of femoral and tibial osteophyte scores for HA andHA+100 million MPC injected joints 12, 24 and 52 weeks postmeniscectomy.

FIG. 9. Ratios [HA/(MPC+HA)] of cartilage morphology joint scores foranimals injected with Mesenchymal Precursor Cells (MPC) 12, 24 and 52weeks post meniscectomy. When ratio=1 both treatments equally effective.Ratios>1 indicate MPC+HA superior to HA.

FIG. 10. Ratios [HA/(MPC+HA)] of osteophyte joint scores for animalsinjected with Mesenchymal Precursor Cells (MPC) 12, 24 and 52 weeks postmeniscectomy. When ratio=1 both treatments equally effective. Ratios>1indicate MPC+HA superior to HA.

FIG. 11. Ratios [HA/(MPC+HA)] of mean±SE Modified Mankins jointcartilage histopathology scores for animals injected with MesenchymalPrecursor Cells (MPC) 12, 24 and 52 weeks post meniscectomy. Whenratios=1 both treatments equally effective. Ratios>1 indicate MPC+HAsuperior to HA.

FIG. 12. Mean±/−SE of patella cartilage stiffness from joints injectedwith hyaluronan (HA) or HA+different doses of Mesenchymal PrecursorCells (MPC). *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001.

FIG. 13. Mean±/−SE of patella cartilage stiffness from joints injectedwith hyaluronan (HA) or 100 million Mesenchymal Precursor Cells (MPC)+HAand sacrificed 12, 24 and 52 weeks post meniscectomy. *=p<0.05,**=p<0.01, ***=p<0.001, ****=p<0.0001.

FIG. 14. Mean±/−SE of patella cartilage phase lag from joints injectedwith hyaluronan (HA) or HA+different doses of Mesenchymal PrecursorCells (MPC). *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001.

FIG. 15. Mean±/−SE of patella cartilage phase lag from joints injectedwith hyaluronan (HA) or HA+100 million Mesenchymal Precursor Cells (MPC)and sacrificed 12, 24 and 52 weeks post meniscectomy. *=p<0.05,**=p<0.01, ***=p<0.001, ****=p<0.0001.

FIG. 16. Comparison of joint cartilage morphology scores for untreatedcastrated male sheep and ovariectomised ewes 12 weeks post meniscectomyshowing the significantly greater severity of OA lesions in the femalegroup.

FIG. 17. Comparison of joint osteophyte scores for untreated castratedmale sheep and ovariectomised ewes 12 weeks post meniscectomy showingthe significantly higher scores in the female group.

FIG. 18. Mean±SD of cartilage Modified Mankin Histopathology scores 36weeks post meniscectomy from joints of ovariectomised ewes injected withHyaluronan (HA) or HA+100 million Mesenchymal Precursor Cells (MPC) 12weeks post meniscectomy. P values=HA versus MPC+HA. These results showthat a single MPC injection reduces abnormal histopathologic score offemoral hyaline cartilage over 6 months to a greater extent than tibialcartilage.

FIG. 19. Ratios (HA/HA+MPC) of cartilage Total Modified MankinHistopathology Scores for joints of ovariectomised ewes 36 weeks postmeniscectomy administered intra-articular injections 12 weeks postmeniscectomy. When ratio=1, MPC+HA equivalent to HA. Ratio>1, showsMPC+HA more protective than HA alone. Data=Means±SEM. These results showthat a single MPC injection reduces abnormal histopathologic score offemoral hyaline cartilage over 6 months to greater extent than tibialcartilage.

FIG. 20. Mean±SD of femoral cartilage Modified Mankin Histopathologyscores 24 and 36 weeks post meniscectomy (MX) from joints ofovariectomised ewes injected with Hyaluronan (HA) or 100 millionMesenchymal Precursor Cells (MPC)+HA 12 weeks post MX compared withnon-injected joints at 12 weeks post MX. P values are for 12 wks NILversus treatments. These results show that a single MPC injectionreduces abnormal histopathologic score over 6 months.

FIG. 21. Femoral cartilage histomorphometry data 36 weeks postmeniscectomy from joints of ovariectomised ewes injected with Hyaluronan(HA) or 100 million Mesenchymal Precursor Cells (MPC)+HA 12 weeks postmeniscectomy. Data shown=Mean±SEM. P values=HA v MPC+HA. These resultsshow that a single MPC injection generates greater hyaline cartilageover 6 Months than hyaluronic acid.

FIG. 22. Mean±SEM histomorphometrically determined femoral cartilagethickness of joints from untreated ewes sacrificed 12 weeks postmeniscectomy (Mx) or injected with Hyaluronan (HA) or MesenchymalPrecursor Cells (MPC)+HA at 12 weeks post Mx then sacrificed 12 or 24weeks later. Data expressed as Mean±SEM. P values relative to 12 weekNIL treated. These results show that a single MPC injection increaseshyaline cartilage thickness over 6 months.

FIG. 23. Histomorphometrically determined femoral cartilage areas ofjoints from untreated ewes sacrificed 12 weeks post meniscectomy (Mx) orinjected with Hyaluronan (HA) or Mesenchymal Precursor Cells (MPC)+HA at12 weeks post Mx then sacrificed 12 or 24 weeks later. Data expressed asMean±SEM. P values relative to 12 week NIL treated. These results showthat a single MPC injection increases hyaline cartilage area over 6months.

FIG. 24. Histomorphometrically determined Integrated Grey-scale Density(IGD) as a measure of overall Proteoglycan (PG) content of femoralcartilages from joints of untreated ewes sacrificed 12 weeks postmeniscectomy (Mx) or injected with Hyaluronan (HA) or MesenchymalPrecursor Cells (MPC)+HA at 12 weeks post Mx then sacrificed 12 or 24weeks later. Data expressed as Mean±SEM. P values relative to 12 weekNIL treated. These results show that a single MPC injection generatessignificantly more cartilage containing proteoglycan than hyaluronicacid injection over 6 months.

FIG. 25. Schematic representation of the lumber spinal levels treatedwith Mesenchymal Precursor Cells (MPC) in all sheep Groups.

FIG. 26. Mean recovery in disc height three and six months followinginjection of MPC and HA into the nuclei pulposi of degenerate sheepdiscs.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION GeneralTechniques and Selected Definitions

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken to have the same meaning as commonlyunderstood by one of ordinary skill in the art (e.g., in cell culture,stem cell biology, molecular genetics, immunology, immunohistochemistry,protein chemistry, and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, andimmunological techniques utilized in the present invention are standardprocedures, well known to those skilled in the art. Such techniques aredescribed and explained throughout the literature in sources such as, J.Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons(1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbour Laboratory Press (1989), T. A. Brown (editor), EssentialMolecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press(1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A PracticalApproach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel etal. (editors), Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience (1988, including all updates untilpresent), Ed Harlow and David Lane (editors) Antibodies: A LaboratoryManual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al.(editors) Current Protocols in Immunology, John Wiley & Sons (includingall updates until present).

As used herein, the terms “treating”, “treat” or “treatment” includeadministering a therapeutically effective amount of supernatant, solublefactors and/or cells as defined herein sufficient to reduce or eliminateat least one symptom of the specified condition.

As used herein, the terms “preventing”, “prevent” or “prevention”include administering a therapeutically effective amount of supernatant,soluble factors and/or cells as defined herein sufficient to stop orhinder the development of at least one symptom of the specifiedcondition.

As used herein, the term “derived from mesenchymal precursor cells”refers to supernatant, and/or one or more soluble factors, produced fromthe in vitro culturing of mesenchymal precursor cells and/or progenycells thereof.

As used herein, the term “supernatant” refers to the non-cellularmaterial produced following the in vitro culturing of mesenchymalprecursor cells, and/or progeny cells thereof, in a suitable medium,preferably liquid medium. Typically, the supernatant is produced byculturing the cells in the medium under suitable conditions and time,followed by removing the cellular material by a process such ascentrifugation. The supernatant may or may not have been subjected tofurther purification steps before administration. In preferredembodiment, the supernatant comprises less than 10⁵, more preferablyless than 10⁴, more preferably less than 10³ and even more preferably nolive cells.

As used herein, the term “one or more soluble” factors refers tomolecules, typically proteins, secreted by the MPCs, and/or progenycells thereof, during culture.

Mesenchymal Precursor Cells (MPCs) or Progeny Cells, and Supernatant orOne or More Soluble Factors Derived Therefrom

As used herein, “MPC” are non-hematopoietic STRO-1⁺ progenitor cellsthat are capable of forming large numbers of multipotential cellcolonies.

Mesenchymal precursor cells (MPCs) are cells found in bone marrow,blood, dental pulp cells, adipose tissue, skin, spleen, pancreas, brain,kidney, liver, heart, retina, brain, hair follicles, intestine, lung,lymph node, thymus, bone, ligament, tendon, skeletal muscle, dermis, andperiosteum; and are capable of differentiating into different germ linessuch as mesoderm, endoderm and ectoderm. Thus, MPCs are capable ofdifferentiating into a large number of cell types including, but notlimited to, adipose, osseous, cartilaginous, elastic, muscular, andfibrous connective tissues. The specific lineage-commitment anddifferentiation pathway which these cells enter depends upon variousinfluences from mechanical influences and/or endogenous bioactivefactors, such as growth factors, cytokines, and/or localmicroenvironmental conditions established by host tissues. Mesenchymalprecursor cells thus non-hematopoietic progenitor cells which divide toyield daughter cells that are either stem cells or are precursor cellswhich in time will irreversibly differentiate to yield a phenotypiccell.

In a preferred embodiment, the MPCs are enriched from a sample obtainedfrom a subject. The terms ‘enriched’, ‘enrichment’ or variations thereofare used herein to describe a population of cells in which theproportion of one particular cell type or the proportion of a number ofparticular cell types is increased when compared with the untreatedpopulation.

In a preferred embodiment, the cells used in the present invention arealso TNAP⁺, VCAM-1⁺, THY-1⁺, STRO-2⁺, CD45⁺, CD146⁺, 3G5⁺ or anycombination thereof. Preferably, the STRO-1⁺ cells are STRO-1^(bright).Preferably, the STRO-1^(bright) cells are additionally one or more ofVCAM-1⁺, THY-1⁺, STRO-2⁺ and/or CD146⁺.

In one embodiment, the mesenchymal precursor cells are perivascularmesenchymal precursor cells as defined in WO 2004/85630.

When we refer to a cell as being “positive” for a given marker it may beeither a low (lo or dim) or a high (bright, bri) expresser of thatmarker depending on the degree to which the marker is present on thecell surface, where the terms relate to intensity of fluorescence orother colour used in the colour sorting process of the cells. Thedistinction of lo (or dim or dull) and bri will be understood in thecontext of the marker used on a particular cell population being sorted.When we refer herein to a cell as being “negative” for a given marker,it does not mean that the marker is not expressed at all by that cell.It means that the marker is expressed at a relatively very low level bythat cell, and that it generates a very low signal when detectablylabelled.

The term “bright”, when used herein, refers to a marker on a cellsurface that generates a relatively high signal when detectablylabelled. Whilst not wishing to be limited by theory, it is proposedthat “bright” cells express more of the target marker protein (forexample the antigen recognised by STRO-1) than other cells in thesample. For instance, STRO-1^(bri) cells produce a greater fluorescentsignal, when labelled with a FITC-conjugated STRO-1 antibody asdetermined by FACS analysis, than non-bright cells (STRO-1^(dull/dim)).Preferably, “bright” cells constitute at least about 0.1% of the mostbrightly labelled bone marrow mononuclear cells contained in thestarting sample. In other embodiments, “bright” cells constitute atleast about 0.1%, at least about 0.5%, at least about 1%, at least about1.5%, or at least about 2%, of the most brightly labelled bone marrowmononuclear cells contained in the starting sample. In a preferredembodiment, STRO-1^(bright) cells have 2 log magnitude higher expressionof STRO-1 surface expression. This is calculated relative to“background”, namely cells that are STRO-1⁻. By comparison, STRO-1^(dim)and/or STRO-1^(intermediate) cells have less than 2 log magnitude higherexpression of STRO-1 surface expression, typically about 1 log or lessthan “background”.

When used herein the term “TNAP” is intended to encompass all isoformsof tissue non-specific alkaline phosphatase. For example, the termencompasses the liver isoform (LAP), the bone isoform (BAP) and thekidney isoform (KAP). In a preferred embodiment, the TNAP is BAP. In aparticularly preferred embodiment, TNAP as used herein refers to amolecule which can bind the STRO-3 antibody produced by the hybridomacell line deposited with ATCC on 19 Dec. 2005 under the provisions ofthe Budapest Treaty under deposit accession number PTA-7282.

Furthermore, in a preferred embodiment, the MPCs are capable of givingrise to clonogenic CFU-F.

It is preferred that a significant proportion of the multipotentialcells are capable of differentiation into at least two different germlines. Non-limiting examples of the lineages to which the multipotentialcells may be committed include bone precursor cells; hepatocyteprogenitors, which are multipotent for bile duct epithelial cells andhepatocytes; neural restricted cells, which can generate glial cellprecursors that progress to oligodendrocytes and astrocytes; neuronalprecursors that progress to neurons; precursors for cardiac muscle andcardiomyocytes, glucose-responsive insulin secreting pancreatic betacell lines. Other lineages include, but are not limited to,odontoblasts, dentin-producing cells and chondrocytes, and precursorcells of the following: retinal pigment epithelial cells, fibroblasts,skin cells such as keratinocytes, dendritic cells, hair follicle cells,renal duct epithelial cells, smooth and skeletal muscle cells,testicular progenitors, vascular endothelial cells, tendon, ligament,cartilage, adipocyte, fibroblast, marrow stroma, cardiac muscle, smoothmuscle, skeletal muscle, pericyte, vascular, epithelial, glial,neuronal, astrocyte and oligodendrocyte cells.

In another embodiment, the MPCs are not capable of giving rise, uponculturing, to hematopoietic cells.

The present invention also relates to use of supernatant or solublefactors obtained derived from MPC and/or progeny cells thereof (thelatter also being referred to as expanded cells) which are produced fromin vitro culture. Expanded cells of the invention may a have a widevariety of phenotypes depending on the culture conditions (including thenumber and/or type of stimulatory factors in the culture medium), thenumber of passages and the like. In certain embodiments, the progenycells are obtained after about 2, about 3, about 4, about 5, about 6,about 7, about 8, about 9, or about 10 passages from the parentalpopulation. However, the progeny cells may be obtained after any numberof passages from the parental population.

The progeny cells may be obtained by culturing in any suitable medium.The term “medium”, as used in reference to a cell culture, includes thecomponents of the environment surrounding the cells. Media may be solid,liquid, gaseous or a mixture of phases and materials. Media includeliquid growth media as well as liquid media that do not sustain cellgrowth. Media also include gelatinous media such as agar, agarose,gelatin and collagen matrices. Exemplary gaseous media include thegaseous phase that cells growing on a petri dish or other solid orsemisolid support are exposed to. The term “medium” also refers tomaterial that is intended for use in a cell culture, even if it has notyet been contacted with cells. In other words, a nutrient rich liquidprepared for bacterial culture is a medium. Similarly, a powder mixturethat when mixed with water or other liquid becomes suitable for cellculture, may be termed a “powdered medium”.

In an embodiment, progeny cells useful for the methods of the inventionare obtained by isolating TNAP+MPCs from bone marrow using magneticbeads labelled with the STRO-3 antibody, and then culture expanding theisolated cells (see Gronthos et al. (1995) for an example of suitableculturing conditions).

In one embodiment, such expanded cells (progeny) (at least after 5passages) can be TNAP-, CC9⁺, HLA class I⁺, HLA class II⁻, CD14⁻, CD19⁻,CD3⁻, CD11a-c⁻, CD31⁻, CD86⁻ CD34⁻ and/or CD80⁻. However, it is possiblethat under different culturing conditions to those described herein thatthe expression of different markers may vary. Also, whilst cells ofthese phenotypes may predominate in the expended cell population it doesnot mean that there is a minor proportion of the cells do not have thisphenotype(s) (for example, a small percentage of the expanded cells maybe CC9−). In one preferred embodiment, expanded cells still have thecapacity to differentiate into different cell types.

In one embodiment, an expended cell population used to obtainsupernatant or soluble factors, or cells per se, comprises cells whereinat least 25%, more preferably at least 50%, of the cells are CC9+.

In another embodiment, an expended cell population used to obtainsupernatant or soluble factors, or cells per se, comprises cells whereinat least 40%, more preferably at least 45%, of the cells are STRO-1+.

In a further embodiment, the expanded cells may express markers selectedfrom the group consisting of LFA-3, THY-1, VCAM-1, ICAM-1, PECAM-1,P-selectin, L-selectin, 3G5, CD49a/CD49b/CD29, CD49c/CD29, CD49d/CD29,CD 90, CD29, CD18, CD61, integrin beta, 6-19, thrombomodulin, CD10,CD13, SCF, PDGF-R, EGF-R, IGF1-R, NGF-R, FGF-R, Leptin-R,(STRO-2=Leptin-R), RANKL, STRO-1^(bright) and CD146 or any combinationof these markers.

In one embodiment, the progeny cells are Multipotential Expanded MPCProgeny (MEMPs) as defined in WO 2006/032092. Methods for preparingenriched populations of MPC from which progeny may be derived aredescribed in WO 01/04268 and WO 2004/085630. In an in vitro context MPCswill rarely be present as an absolutely pure preparation and willgenerally be present with other cells that are tissue specific committedcells (TSCCs). WO 01/04268 refers to harvesting such cells from bonemarrow at purity levels of about 0.1% to 90%. The population comprisingMPC from which progeny are derived may be directly harvested from atissue source, or alternatively it may be a population that has alreadybeen expanded ex vivo.

For example, the progeny may be obtained from a harvested, unexpanded,population of substantially purified MPC, comprising at least about 0.1,1, 5, 10, 20, 30, 40, 50, 60, 70, 80 or 95% of total cells of thepopulation in which they are present. This level may be achieved, forexample, by selecting for cells that are positive for at least onemarker selected from the group consisting of TNAP, STRO-1^(bright),3G5⁺, VCAM-1, THY-1, CD146 and STRO-2.

MEMPS can be distinguished from freshly harvested MPCs in that they arepositive for the marker STRO-1^(bri) and negative for the markerAlkaline phosphatase (ALP). In contrast, freshly isolated MPCs arepositive for both STRO-1^(bri) and ALP. In a preferred embodiment of thepresent invention, at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%or 95% of the administered cells have the phenotype STRO-1^(bri), ALP⁻.In a further preferred embodiment the MEMPS are positive for one or moreof the markers Ki67, CD44 and/or CD49c/CD29, VLA-3, α3β1. In yet afurther preferred embodiment the MEMPs do not exhibit TERT activityand/or are negative for the marker CD18.

The MPC starting population may be derived from any one or more tissuetypes set out in WO 01/04268 or WO 2004/085630, namely bone marrow,dental pulp cells, adipose tissue and skin, or perhaps more broadly fromadipose tissue, teeth, dental pulp, skin, liver, kidney, heart, retina,brain, hair follicles, intestine, lung, spleen, lymph node, thymus,pancreas, bone, ligament, bone marrow, tendon and skeletal muscle.

It will be understood that in performing the present invention,separation of cells carrying any given cell surface marker can beeffected by a number of different methods, however, preferred methodsrely upon binding a binding agent to the marker concerned followed by aseparation of those that exhibit binding, being either high levelbinding, or low level binding or no binding. The most convenient bindingagents are antibodies or antibody based molecules, preferably beingmonoclonal antibodies or based on monoclonal antibodies because of thespecificity of these latter agents. Antibodies can be used for bothsteps, however other agents might also be used, thus ligands for thesemarkers may also be employed to enrich for cells carrying them, orlacking them.

The antibodies or ligands may be attached to a solid support to allowfor a crude separation. The separation techniques preferably maximisethe retention of viability of the fraction to be collected. Varioustechniques of different efficacy may be employed to obtain relativelycrude separations. The particular technique employed will depend uponefficiency of separation, associated cytotoxicity, ease and speed ofperformance, and necessity for sophisticated equipment and/or technicalskill. Procedures for separation may include, but are not limited to,magnetic separation, using antibody-coated magnetic beads, affinitychromatography and “panning” with antibody attached to a solid matrix.Techniques providing accurate separation include but are not limited toFACS.

It is preferred that the method for isolating MPCs, for example,comprises a first step being a solid phase sorting step utilising forexample MACS recognising high level expression of STRO-1. A secondsorting step can then follow, should that be desired, to result in ahigher level of precursor cell expression as described in patentspecification WO 01/14268. This second sorting step might involve theuse of two or more markers.

The method obtaining MPCs might also include the harvesting of a sourceof the cells before the first enrichment step using known techniques.Thus the tissue will be surgically removed. Cells comprising the sourcetissue will then be separated into a so called single cells suspension.This separation may be achieved by physical and or enzymatic means.

Once a suitable MPC population has been obtained, it may be cultured orexpanded by any suitable means to obtain MEMPs.

In one embodiment, the cells are taken from the subject to be treated,cultured in vitro using standard techniques and used to obtainsupernatant or soluble factors or expanded cells for administration tothe subject as an autologous or allogeneic composition. In analternative embodiment, cells of one or more of the established humancell lines are used to obtain the supernatant or soluble factors. Inanother useful embodiment of the invention, cells of a non-human animal(or if the patient is not a human, from another species) are used toobtain supernatant or soluble factors.

The invention can be practised using cells from any non-human animalspecies, including but not limited to non-human primate cells, ungulate,canine, feline, lagomorph, rodent, avian, and fish cells. Primate cellswith which the invention may be performed include but are not limited tocells of chimpanzees, baboons, cynomolgus monkeys, and any other New orOld World monkeys. Ungulate cells with which the invention may beperformed include but are not limited to cells of bovines, porcines,ovines, caprines, equines, buffalo and bison. Rodent cells with whichthe invention may be performed include but are not limited to mouse,rat, guinea pig, hamster and gerbil cells. Examples of lagomorph specieswith which the invention may be performed include domesticated rabbits,jack rabbits, hares, cottontails, snowshoe rabbits, and pikas. Chickens(Gallus gallus) are an example of an avian species with which theinvention may be performed.

Cells useful for the methods of the invention may be stored before use,or before obtaining the supernatant or soluble factors. Methods andprotocols for preserving and storing of eukaryotic cells, and inparticular mammalian cells, are well known in the art (cf., for example,Pollard, J. W. and Walker, J. M. (1997) Basic Cell Culture Protocols,Second Edition, Humana Press, Totowa, N.J.; Freshney, R. I. (2000)Culture of Animal Cells, Fourth Edition, Wiley-Liss, Hoboken, N.J.). Anymethod maintaining the biological activity of the isolated stem cellssuch as mesenchymal stem/progenitor cells, or progeny thereof, may beutilized in connection with the present invention. In one preferredembodiment, the cells are maintained and stored by usingcryo-preservation.

Administration and Compositions Supernatant or Soluble Factors

The methods of the present invention may involve administeringMPC-derived supernatant or soluble factors, topically, systematically,or locally such as within an implant or device.

In one particular embodiment the invention involves administeringMPC-derived supernatant or soluble factors systemically to the subject.For example, the supernatant or soluble factors may be administered bysubcutaneous or intramuscular injection.

This embodiment of the invention may be useful for the treatment ofsystemic degenerative diseases where generation or repair of particulartissues is desirable. Examples of systemic degenerative diseases thatcan be treated in this way include osteoporosis or fractures, ordegenerative diseases of cartilage.

The MPC-derived supernatant or soluble factors may also be used to treatpatients requiring the repair or replacement of cartilage tissueresulting from disease or trauma or failure of the tissue to developnormally, or to provide a cosmetic function, such as to augment facialor other features of the body. Treatment may entail the use of thesupernatant or soluble factors to produce new cartilage tissue and/ormaintain existing cartilage tissue. For example, MPC-derived supernatantor soluble factors may be used to treat a cartilage condition, forexample, rheumatoid arthritis or osteoarthritis or a traumatic orsurgical injury to cartilage.

Suspensions comprising MPC-derived supernatant or soluble factors may beprepared as appropriate oily suspensions for injection. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil;or synthetic fatty acid esters, such as ethyl oleate or triglycerides;or liposomes. Suspensions to be used for injection may also containsubstances which increase the viscosity of the suspension, such assodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, thesuspension may also contain suitable stabilizers or agents whichincrease the solubility of the compounds to allow for the preparation ofhighly concentrated solutions.

Sterile injectable solutions can be prepared by incorporating thesupernatant or soluble factors in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the supernatant or soluble factors into asterile vehicle that contains a basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.In accordance with an alternative aspect of the invention, thesupernatant or soluble factors may be formulated with one or moreadditional compounds that enhance its solubility.

Cellular Compositions

In one embodiment, cellular compositions of the invention areadministered as undifferentiated cells, i.e., as cultured in GrowthMedium. Alternatively, the cellular compositions may be administeredfollowing culturing.

The cellular compositions useful for the present invention may beadministered alone or as admixtures with other cells. Cells that may beadministered in conjunction with the compositions of the presentinvention include, but are not limited to, other multipotent orpluripotent cells or chondrocytes, chondroblasts, osteocytes,osteoblasts, osteoclasts, bone lining cells, stem cells, or bone marrowcells. The cells of different types may be admixed with a composition ofthe invention immediately or shortly prior to administration, or theymay be co-cultured together for a period of time prior toadministration.

In some embodiments of the invention, it may not be necessary ordesirable to immunosuppress a patient prior to initiation of therapywith cellular compositions. Accordingly, transplantation withallogeneic, or even xenogeneic, MPCs or progeny thereof may be toleratedin some instances.

However, in other instances it may be desirable or appropriate topharmacologically immunosuppress a patient prior to initiating celltherapy. This may be accomplished through the use of systemic or localimmunosuppressive agents, or it may be accomplished by delivering thecells in an encapsulated device. The cells may be encapsulated in acapsule that is permeable to nutrients and oxygen required by the celland therapeutic factors the cell is yet impermeable to immune humoralfactors and cells. Preferably the encapsulant is hypoallergenic, iseasily and stably situated in a target tissue, and provides addedprotection to the implanted structure. These and other means forreducing or eliminating an immune response to the transplanted cells areknown in the art. As an alternative, the cells may be geneticallymodified to reduce their immunogenicity.

General

A “therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredeffect.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result, such as preventing or inhibiting cell apoptosis ortissue damage.

The amount of supernatant or soluble factors, or MPCs or progeny thereofto be administered may vary according to factors such as the diseasestate, age, sex, and weight of the individual. Dosage regimens may beadjusted to provide the optimum therapeutic response. For example, asingle bolus may be administered, several divided doses may beadministered over time or the dose may be proportionally reduced orincreased as indicated by the exigencies of the therapeutic situation.It may be advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. “Dosageunit form” as used herein refers to physically discrete units suited asunitary dosages for subjects to be treated; each unit containing apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier.

It will be appreciated that the supernatant or soluble factors or MPCsor progeny thereof may be administered in the form of a compositioncomprising a pharmaceutically acceptable carrier or excipient.

As used herein “pharmaceutically acceptable carrier” or “excipient”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike that are physiologically compatible. In one embodiment, the carrieris suitable for parenteral administration. Alternatively, the carriercan be suitable for intravenous, intraperitoneal, intramuscular,sublingual or oral administration. Pharmaceutically acceptable carriersinclude sterile aqueous solutions or dispersions and sterile, powdersfor the extemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe pharmaceutical compositions of the invention is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

Therapeutic compositions typically should be sterile and stable underthe conditions of manufacture and storage. The composition can beformulated as a solution, microemulsion, liposome, or other orderedstructure. The carrier can be a solvent or dispersion medium containing,for example, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. In many cases, it will be preferable to include isotonicagents, for example, sugars, polyalcohols such as mannitol, sorbitol, orsodium chloride in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, monostearatesalts and gelatin. Moreover, the stimulatory factor may be administeredin a time release formulation, for example in a composition whichincludes a slow release polymer. The active compounds can be preparedwith carriers that will protect the compound against rapid release, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, polylactic acid andpolylactic, polyglycolic copolymers (PLG). Many methods for thepreparation of such formulations are patented or generally known tothose skilled in the art.

The supernatant or soluble factors or cell compositions may beadministered in combination with an appropriate matrix, for instance, toprovide slow release of the soluble factors.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. Potential matrices for the compositions may bebiodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid and polyanhydrides. Otherpotential materials are biodegradable and biologically well defined,such as bone or dermal collagen. Further matrices are comprised of pureproteins or extracellular matrix components. Other potential matricesare nonbiodegradable and chemically defined, such as sinteredhydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may becomprised of combinations of any of the above mentioned types ofmaterial, such as polylactic acid and hydroxyapatite or collagen andtricalcium phosphate. The bioceramics may be altered in composition,such as in calcium-aluminate-phosphate and processing to alter poresize, particle size, particle shape, and biodegradability.

The MPC-derived supernatant or soluble factors, MPCs or progeny thereofmay be surgically implanted, injected, delivered (e.g., by way of acatheter or syringe), or otherwise administered directly or indirectlyto the site in need of repair or augmentation. Routes of administrationof the MPC-derived supernatant or soluble factors include intramuscular,ophthalmic, parenteral (including intravenous), intraarterial,subcutaneous, oral, and nasal administration. Particular routes ofparenteral administration include, but are not limited to,intramuscular, subcutaneous, intraperitoneal, intracerebral,intraventricular, intracerebroventricular, intrathecal, intracisternal,intraspinal and/or peri-spinal routes of administration.

In some embodiments of the invention, the formulation comprises an insitu polymerizable gel, as described, for example, in US 2002/0022676;Anseth et al. (2002) and Wang et al. (2003).

In some embodiments, the polymers are at least partially soluble inaqueous solutions, such as water, buffered salt solutions, or aqueousalcohol solutions, that have charged side groups, or a monovalent ionicsalt thereof. Examples of polymers with acidic side groups that can bereacted with cations are poly(phosphazenes), poly(acrylic acids),poly(methacrylic acids), copolymers of acrylic acid and methacrylicacid, poly(vinyl acetate), and sulfonated polymers, such as sulfonatedpolystyrene. Copolymers having acidic side groups formed by reaction ofacrylic or methacrylic acid and vinyl ether monomers or polymers canalso be used. Examples of acidic groups are carboxylic acid groups,sulfonic acid groups, halogenated (preferably fluorinated) alcoholgroups, phenolic OH groups, and acidic OH groups.

Examples of polymers with basic side groups that can be reacted withanions are poly(vinyl amines), poly(vinyl pyridine), poly(vinylimidazole), and some imino substituted polyphosphazenes. The ammonium orquaternary salt of the polymers can also be formed from the backbonenitrogens or pendant imino groups. Examples of basic side groups areamino and imino groups.

Alginate can be ionically cross-linked with divalent cations, in water,at room temperature, to form a hydrogel matrix. Due to these mildconditions, alginate has been the most commonly used polymer forhybridoma cell encapsulation, as described, for example, in U.S. Pat.No. 4,352,883. In the process described in U.S. Pat. No. 4,352,883, anaqueous solution containing the biological materials to be encapsulatedis suspended in a solution of a water soluble polymer, the suspension isformed into droplets which are configured into discrete microcapsules bycontact with multivalent cations, then the surface of the microcapsulesis crosslinked with polyamino acids to form a semipermeable membranearound the encapsulated materials.

Polyphosphazenes are polymers with backbones consisting of nitrogen andphosphorous separated by alternating single and double bonds. Eachphosphorous atom is covalently bonded to two side chains.

The polyphosphazenes suitable for cross-linking have a majority of sidechain groups which are acidic and capable of forming salt bridges withdi- or trivalent cations. Examples of preferred acidic side groups arecarboxylic acid groups and sulfonic acid groups. Hydrolytically stablepolyphosphazenes are formed of monomers having carboxylic acid sidegroups that are crosslinked by divalent or trivalent cations such asCa²⁺ or Al³⁺. Polymers can be synthesized that degrade by hydrolysis byincorporating monomers having imidazole, amino acid ester, or glycerolside groups. For example, a polyanionicpoly[bis(carboxylatophenoxy)]phosphazene (PCPP) can be synthesized,which is cross-linked with dissolved multivalent cations in aqueousmedia at room temperature or below to form hydrogel matrices.

Biodegradable polyphosphazenes have at least two differing types of sidechains, acidic side groups capable of forming salt bridges withmultivalent cations, and side groups that hydrolyze under in vivoconditions, e.g., imidazole groups, amino acid esters, glycerol andglucosyl.

Hydrolysis of the side chain results in erosion of the polymer. Examplesof hydrolyzing side chains are unsubstituted and substituted imidizolesand amino acid esters in which the group is bonded to the phosphorousatom through an amino linkage (polyphosphazene polymers in which both Rgroups are attached in this manner are known as polyaminophosphazenes).For polyimidazolephosphazenes, some of the “R” groups on thepolyphosphazene backbone are imidazole rings, attached to phosphorous inthe backbone through a ring nitrogen atom. Other “R” groups can beorganic residues that do not participate in hydrolysis, such as methylphenoxy groups or other groups shown in the scientific paper of Allcocket al. (1977). Methods of synthesis of the hydrogel materials, as wellas methods for preparing such hydrogels, are known in the art.

The MPC-derived supernatant or soluble factors, MPCs or progeny thereofmay be administered with other beneficial drugs or biological molecules(growth factors, trophic factors). When administered with other agents,they may be administered together in a single pharmaceuticalcomposition, or in separate pharmaceutical compositions, simultaneouslyor sequentially with the other agents (either before or afteradministration of the other agents). Bioactive factors which may beco-administered include anti-apoptotic agents (e.g., EPO, EPOmimetibody, TPO, IGF-I and IGF-II, HGF, caspase inhibitors);anti-inflammatory agents (e.g., p38 MAPK inhibitors, TGF-betainhibitors, statins, IL-6 and IL-1 inhibitors, PEMIROLAST, TRANILAST,REMICADE, SIROLIMUS, and NSAIDs (non-steroidal anti-inflammatory drugs;e.g., TEPDXALIN, TOLMETIN, SUPROFEN); immunosupressive/immunomodulatoryagents (e.g., calcineurin inhibitors, such as cyclosporine, tacrolimus;mTOR inhibitors (e.g., SIROLIMUS, EVEROLIMUS); anti-proliferatives(e.g., azathioprine, mycophenolate mofetil); corticosteroids (e.g.,prednisolone, hydrocortisone); antibodies such as monoclonalanti-IL-2Ralpha receptor antibodies (e.g., basiliximab, daclizumab),polyclonal anti-T-cell antibodies (e.g., anti-thymocyte globulin (ATG);anti-lymphocyte globulin (ALG); monoclonal anti-T cell antibody OKT3));anti-thrombogenic agents (e.g., heparin, heparin derivatives, urokinase,PPack (dextrophenylalanine proline arginine chloromethylketone),antithrombin compounds, platelet receptor antagonists, anti-thrombinantibodies, anti-platelet receptor antibodies, aspirin, dipyridamole,protamine, hirudin, prostaglandin inhibitors, and platelet inhibitors);and anti-oxidants (e.g., probucol, vitamin A, ascorbic acid, tocopherol,coenzyme Q-10, glutathione, L-cysteine, N-acetylcysteine) as well aslocal anesthetics. As another example, the MPC-derived supernatant orsoluble factors, MPCs or progeny thereof may be co-administered withscar inhibitory factor as described in U.S. Pat. No. 5,827,735.

When treating and/or preventing a disease arising from degradationand/or inflammation of connective tissue it is preferred that thesupernatant, soluble factors or cells are administered withchondroprotective agents. Examples include, but are not limited to,pentosan polysulfate (SP54 and Cartrophen), glycosaminoglycan polysufateester (Arteparon), glyciamino-glycan-peptide complex (Rumalon) andhyaluronic acid (Hyalgan). Further examples are described by Verbruggen(2005) and Richette and Bardin (2004). In a preferred embodiment, thechondroprotective agent is hyaluronic acid.

Fibrin Glue

Fibrin glues are a class of surgical sealants which have been used invarious clinical settings. As the skilled address would be aware,numerous sealants are useful for the methods defined herein. However, apreferred embodiment of the invention relates to the use of fibringlues.

When used herein the term “fibrin glue” refers to the insoluble matrixformed by the cross-linking of fibrin polymers in the presence ofcalcium ions. The fibrin glue may be formed from fibrinogen, or aderivative or metabolite thereof, fibrin (soluble monomers or polymers)and/or complexes thereof derived from biological tissue or fluid whichforms a fibrin matrix. Alternatively, the fibrin glue may be formed fromfibrinogen, or a derivative or metabolite thereof, or fibrin, producedby recombinant DNA technology.

The fibrin glue may also be formed by the interaction of fibrinogen anda catalyst of fibrin glue formation (such as thrombin and/or FactorXIII). As will be appreciated by those skilled in the art, fibrinogen isproteolytically cleaved in the presence of a catalyst (such as thrombin)and converted to a fibrin monomer. The fibrin monomers may then formpolymers which may cross-link to form a fibrin glue matrix. Thecross-linking of fibrin polymers may be enhanced by the presence of acatalyst such as Factor XIII. The catalyst of fibrin glue formation maybe derived from blood plasma, cryoprecipitate or other plasma fractionscontaining fibrinogen or thrombin. Alternatively, the catalyst may beproduced by recombinant DNA technology.

The rate at which the clot forms is dependent upon the concentration ofthrombin mixed with fibrinogen. Being an enzyme dependent reaction, thehigher the temperature (up to 37° C.) the faster the clot formationrate. The tensile strength of the clot is dependent upon theconcentration of fibrinogen used.

Use of fibrin glue and methods for its preparation and use are describedby Hirsh et al. in U.S. Pat. No. 5,643,192. Hirsh discloses theextraction of fibrinogen and thrombin components from a single donor,and the combination of only these components for use as a fibrin glue.Marx, U.S. Pat. No. 5,651,982, describes another preparation and methodof use for fibrin glue. Marx provides a fibrin glue with liposomes foruse as a topical sealant in mammals. The preparation and use of atopical fibrinogen complex (TFC) for wound healing is known in thefield. International Patent Publication No. WO96/17633, of The AmericanRed Cross, discusses TFC preparations containing fibrinogen, thrombin,and calcium chloride.

Several publications describe the use of fibrin glue for the delivery oftherapeutic agents. For example, U.S. Pat. No. 4,983,393 discloses acomposition for use as an intra-vaginal insert comprising agarose, agar,saline solution glycosaminoglycans, collagen, fibrin and an enzyme.Further, U.S. Pat. No. 3,089,815 discloses an injectable pharmaceuticalpreparation composed of fibrinogen and thrombin and U.S. Pat. No.6,468,527 discloses a fibrin glue which facilitates the delivery ofvarious biological and non-biological agents to specific sites withinthe body.

Production of Genetically Modified Cells

In one embodiment, the cells used in the methods of the invention,including for the production of supernatant or soluble factors, aregenetically modified. Preferably, the cells are genetically modified toproduce a heterologous protein. Typically, the cells will be geneticallymodified such that the heterologous protein is secreted from the cells.However, in an embodiment the cells can be modified to express afunctional non-protein encoding polynucleotide such as dsRNA (typicallyfor RNA silencing), an antisense oligonucleotide or a catalytic nucleicacid (such as a ribozyme or DNAzyme).

Genetically modified cells may be cultured in the presence of at leastone cytokine in an amount sufficient to support growth of the modifiedcells. The genetically modified cells thus obtained may be usedimmediately (e.g., in transplant), cultured and expanded in vitro, orstored for later uses. The modified cells may be stored by methods wellknown in the art, e.g., frozen in liquid nitrogen.

Genetic modification as used herein encompasses any genetic modificationmethod which involves introduction of an exogenous or foreignpolynucleotide into a cell described herein or modification of anendogenous gene within the cell. Genetic modification includes but isnot limited to transduction (viral mediated transfer of host DNA from ahost or donor to a recipient, either in vitro or in vivo), transfection(transformation of cells with isolated viral DNA genomes), liposomemediated transfer, electroporation, calcium phosphate transfection orcoprecipitation and others. Methods of transduction include directco-culture of cells with producer cells (Bregni et al., 1992) orculturing with viral supernatant alone with or without appropriategrowth factors and polycations.

An exogenous polynucleotide is preferably introduced to the cell in avector. The vector preferably includes the necessary elements for thetranscription and translation of the inserted coding sequence. Methodsused to construct such vectors are well known in the art. For example,techniques for constructing suitable expression vectors are described indetail in Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press, N.Y. (3rd Ed., 2000); and Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc., New York(1999).

Vectors may include, but are not limited to, viral vectors, such asretroviruses, adenoviruses, adeno-associated viruses, and herpes simplexviruses; cosmids; plasmid vectors; synthetic vectors; and otherrecombination vehicles typically used in the art. Vectors containingboth a promoter and a cloning site into which a polynucleotide can beoperatively linked are well known in the art. Such vectors are capableof transcribing RNA in vitro or in vivo, and are commercially availablefrom sources such as Stratagene (La Jolla, Calif.) and Promega Biotech(Madison, Wis.). Specific examples include, pSG, pSV2CAT, pXtl fromStratagene; and pMSG, pSVL, pBPV and pSVK3 from Pharmacia.

Preferred vectors include retroviral vectors (see, Coffin et al.,“Retroviruses”, Chapter 9 pp; 437-473, Cold Springs Harbor LaboratoryPress, 1997). Vectors useful in the invention can be producedrecombinantly by procedures well known in the art. For example,WO94/29438, WO97/21824 and WO97/21825 describe the construction ofretroviral packaging plasmids and packing cell lines. Exemplary vectorsinclude the pCMV mammalian expression vectors, such as pCMV6b and pCMV6c(Chiron Corp.), pSFFV-Neo, and pBluescript-Sk+. Non-limiting examples ofuseful retroviral vectors are those derived from murine, avian orprimate retroviruses. Common retroviral vectors include those based onthe Moloney murine leukemia virus (MoMLV-vector). Other MoMLV derivedvectors include, Lmily, LINGFER, MINGFR and MINT. Additional vectorsinclude those based on Gibbon ape leukemia virus (GALV) and Moloneymurine sarcoma virus (MOMSV) and spleen focus forming virus (SFFV).Vectors derived from the murine stem cell virus (MESV) includeMESV-MiLy. Retroviral vectors also include vectors based onlentiviruses, and non-limiting examples include vectors based on humanimmunodeficiency virus (HIV-1 and HIV-2).

In producing retroviral vector constructs, the viral gag, pol and envsequences can be removed from the virus, creating room for insertion offoreign DNA sequences. Genes encoded by foreign DNA are usuallyexpressed under the control a strong viral promoter in the long terminalrepeat (LTR). Selection of appropriate control regulatory sequences isdependent on the host cell used and selection is within the skill of onein the art. Numerous promoters are known in addition to the promoter ofthe LTR. Non-limiting examples include the phage lambda PL promoter, thehuman cytomegalovirus (CMV) immediate early promoter; the U3 regionpromoter of the Moloney Murine Sarcoma Virus (MMSV), Rous Sacroma Virus(RSV), or Spleen Focus Forming Virus (SFFV); Granzyme A promoter; andthe Granzyme B promoter. Additionally inducible or multiple controlelements may be used. The selection of a suitable promoter will beapparent to those skilled in the art.

Such a construct can be packed into viral particles efficiently if thegag, pol and env functions are provided in trans by a packing cell line.Therefore, when the vector construct is introduced into the packagingcell, the gag-pol and env proteins produced by the cell, assemble withthe vector RNA to produce infectious virons that are secreted into theculture medium. The virus thus produced can infect and integrate intothe DNA of the target cell, but does not produce infectious viralparticles since it is lacking essential packaging sequences. Most of thepacking cell lines currently in use have been transfected with separateplasmids, each containing one of the necessary coding sequences, so thatmultiple recombination events are necessary before a replicationcompetent virus can be produced. Alternatively the packaging cell lineharbours a provirus. The provirus has been crippled so that although itmay produce all the proteins required to assemble infectious viruses,its own RNA cannot be packaged into virus. RNA produced from therecombinant virus is packaged instead. Therefore, the virus stockreleased from the packaging cells contains only recombinant virus.Non-limiting examples of retroviral packaging lines include PA12, PA317,PE501, PG13, PSI.CRIP, RDI 14, GP7C-tTA-G10, ProPak-A (PPA-6), and PT67.

Other suitable vectors include adenoviral vectors (see, WO 95/27071) andadeno-associated viral vectors. These vectors are all well known in theart, e.g., as described in Stem Cell Biology and Gene Therapy, eds.Quesenberry et al., John Wiley & Sons, 1998; and U.S. Pat. Nos.5,693,531 and 5,691,176. The use of adenovirus-derived vectors may beadvantageous under certain situation because they are not capable ofinfecting non-dividing cells. Unlike retroviral DNA, the adenoviral DNAis not integrated into the genome of the target cell. Further, thecapacity to carry foreign DNA is much larger in adenoviral vectors thanretroviral vectors. The adeno-associated viral vectors are anotheruseful delivery system. The DNA of this virus may be integrated intonon-dividing cells, and a number of polynucleotides have been successfulintroduced into different cell types using adeno-associated viralvectors.

In some embodiments, the construct or vector will include two or moreheterologous polynucleotide sequences. Preferably the additional nucleicacid sequence is a polynucleotide which encodes a selective marker, astructural gene, a therapeutic gene, or a cytokine/chemokine gene.

A selective marker may be included in the construct or vector for thepurposes of monitoring successful genetic modification and for selectionof cells into which DNA has been integrated. Non-limiting examplesinclude drug resistance markers, such as G148 or hygromycin.Additionally negative selection may be used, for example wherein themarker is the HSV-tk gene. This gene will make the cells sensitive toagents such as acyclovir and gancyclovir. The NeoR (neomycin/G148resistance) gene is commonly used but any convenient marker gene may beused whose gene sequences are not already present in the target cell canbe used. Further non-limiting examples include low-affinity Nerve GrowthFactor (NGFR), enhanced fluorescent green protein (EFGP), dihydrofolatereductase gene (DHFR) the bacterial hisD gene, murine CD24 (HSA), murineCD8a(lyt), bacterial genes which confer resistance to puromycin orphleomycin, and β-glactosidase.

The additional polynucleotide sequence(s) may be introduced into thecell on the same vector or may be introduced into the host cells on asecond vector. In a preferred embodiment, a selective marker will beincluded on the same vector as the polynucleotide.

The present invention also encompasses genetically modifying thepromoter region of an endogenous gene such that expression of theendogenous gene is up-regulated resulting in the increased production ofthe encoded protein compared to a wild type cell.

EXAMPLES Example 1 Expansion of Immunoselected MPCs and Collection ofSupernatant

Bone marrow (BM) is harvested from sheep less than 2 years old. Briefly,40 ml of BM is aspirated from the anterior iliac crest intolithium-heparin anticoagulant-containing tubes. BMMNC are prepared bydensity gradient separation using Lymphoprep™ (Nycomed Pharma, Oslo,Norway) as previously described (Zannettino et al., 1998). Followingcentrifugation at 400×g for 30 minutes at 4° C., the buffy layer isremoved with a transfer pipette and washed three times in “HHF”,composed of Hank's balanced salt solution (HBSS; Life Technologies,Gaithersburg, Md.), containing 5% fetal calf serum (FCS, CSL Limited,Victoria, Australia).

TNAP+ were subsequently isolated by magnetic activated cell sorting aspreviously described (Gronthos et al., 2003; Gronthos et al., 1995).Briefly, approximately 1-3×10⁸ BMMNC are incubated in blocking buffer,consisting of 10% (v/v) normal rabbit serum in HHF for 20 minutes onice. The cells are incubated with 200 μl of a 10 μg/ml solution ofSTRO-3 mAb in blocking buffer for 1 hour on ice. The cells aresubsequently washed twice in HHF by centrifugation at 400×g. A 1/50dilution of goat anti-mouse γ-biotin (Southern Biotechnology Associates,Birmingham, UK) in HHF buffer is added and the cells incubated for 1hour on ice. Cells are washed twice in MACS buffer (Ca²⁺- and Mn²⁺-freePBS supplemented with 1% BSA, 5 mM EDTA and 0.01% sodium azide) as aboveand resuspended in a final volume of 0.9 ml MACS buffer.

One hundred μl streptavidin microbeads (Miltenyi Biotec; BergischGladbach, Germany) are added to the cell suspension and incubated on icefor 15 minutes. The cell suspension is washed twice and resuspended in0.5 ml of MACS buffer and subsequently loaded onto a mini MACS column(MS Columns, Miltenyi Biotec), and washed three times with 0.5 ml MACSbuffer to retrieve the cells which did not bind the STRO-3 mAb(deposited on 19 Dec. 2005 with American Type Culture Collection (ATCC)under accession number PTA-7282—see WO/2006/108229). After addition of afurther 1 ml MACS buffer, the column is removed from the magnet and theTNAP-positive cells are isolated by positive pressure. An aliquot ofcells from each fraction can be stained with streptavidin-FITC and thepurity assessed by flow cytometry.

Primary cultures are established from the MACS isolated TNAP+ cells byplating in α-MEM supplemented with 20% fetal calf serum, 2 mML-glutamine and 100 μm L-ascorbate-2-phosphate as previously described(Gronthos et al., 1995).

Cells were cultured up to passage 5 at which point the conditionedmedium (supernatant) may be collected.

Example 2 Studies on the Dose Dependent Intra-Articular Effects ofAllogeneic Immunoselected Mesenchymal Precursors Cells (MPC) onCartilage Integrity in a Model of Early OA Induced by Bilateral TotalMedial Meniscectomy in Adult Castrated Male Sheep (Wethers)

The knee joint menisci, or semi-lunar cartilages, are important weightbearing structures that also serve to improve articular cartilagelubrication and provide lateral stabilization during joint articulation.Surgical removal of a torn or degenerate meniscus, i.e., meniscectomy,is a common orthopaedic procedure but is known to be associated with anincreased risk of osteoarthritis (OA) in later years (Englund, 2004).Mechanical entrapment of the joint synovium in the space previouslyoccupied by the surgically excised meniscus is known to lead to thepartial regeneration of a meniscus replica (Moon et al., 1984). However,the results of experimental meniscectomy studies in dogs indicate thatthese replacement structures consisted essentially of fibrous tissuewith far inferior biomechanical properties to the original menisci(Ghosh et al., 1983). Furthermore, the extent of OA development in thejoints of these experimental animals 6 months post-meniscectomy wasrelatively severe, confirming the limited functional protection offeredby the regrown structures on articular cartilage (Ghosh et al. 1983a).Large and small animal models of OA have permitted longitudinalevaluation of spatial and temporal changes in joint tissues that occurduring the development of this disease which is difficult obtain usinghuman patients (Smith and Ghosh, 2001). In merino sheep, lateral ormedial meniscectomy has been shown to reliably reproduce biochemical,biomechanical and histopathological alterations typical of OA (Smith andGhosh, 2001). The ovine OA model has also been extensively used toinvestigate the outcomes of various modalities of post-operativetreatments (Ghosh, 1991; Smith and Ghosh, 2001) but to date has not beenemployed to evaluate meniscal regrowth and the progression of OA and howthese events might be influenced by intra-articular mesenchymalprecursor cell (MPC) therapy.

Our previous studies had shown that Bilateral Total Medial Meniscectomy(BTM) in merino sheep resulted in pathological changes in articularcartilage (AC), subchondral bone and synovial tissues that wereprogressive and simulated the development of early human osteoarthritis(OA). We previously used this animal model to evaluate potentialdisease-modifying OA drugs.

Methods

BTM was undertaken in 36 adult Merino wethers. Two weeks post BTM,joints were randomly injected with either 2 mL high MW Hyaluronan (HA)or 2 mL allogeneic Stro-3+ MPC suspended in 2 mL HA. Four doses of MPCwere studied: Group A=10 million (mil) MPC [n=6]; Group B=25 mil MPC[n=6]; Group C=100 mil MPC [n=18] and Group D=150 mil MPC [n=6]. GroupsA, B and D were sacrificed 12 weeks post-BTM while Group C weresacrificed 12 [n=6], 24 [n=6] and 52 [n=6] weeks post-BTM.

At necropsy, both medial compartments of BTM joints were scored by 2blinded observers for AC lesions and osteophytes (OP) using a 0-4 scale.Synovial tissue and a 5 mm wide coronal osteochondral slice were removedfrom the mid-line of the femur and tibia and processed and scored forhistopathological changes (Little et al., 1997) and histomorphometricanalyses (Cake et al., 2003) using the methods cited.

Intact patellae from all joints were subjected to topographicalbiomechanical indentation studies to deterine the stiffness and phaselag of the articular cartilage (Appleyard et al., 2003).

Statistical analysis for treatment effect was undertaken usingKruskal-Wallis nonparametric analysis and for specific between groupcomparisons using Mann Whitney U nonparametric analysis with p<0.05considered significant.

Statistical analysis for comparison between group means for MPC+HAinjected and HA injected joints of each group was undertaken using theequal variance two tailed Student's T-Test with p<0.05 consideredsignificant.

Statistical analysis for comparison between patella cartilages fromMPC+HA injected (Treated) and HA injected joints of each group wasundertaken using an independent T-Test with p<0.05 consideredsignificant.

Results

Gross morphological scores 12 wks post BTM showed a dose-dependenteffect of MPC on AC integrity and OP formation; 100 mil MPC emerging asthe most effective chondroprotective dose relative to HA alone (FIGS. 1and 2). Total AC score ratios (HA+MPC)/(HA) showed 100>150>25=10 whileOP ratios were 100=25>10>150 mil MPC (FIGS. 3 and 4). Statisticallysignificant (SS) lower score were observed for total femoral & tibial AC(p=0.02) while p=0.052 was observed for Group C MPC femoral cartilagescompared to HA alone (FIG. 1).

Histomorphometric analysis of Group C MPC+HA tibial plateau revealedthat AC was thicker than the corresponding HA-AC in the middle (p=0.057)and outer regions (p=0.028); all regions (p=0.01) (FIG. 5). Meanmodified Mankin scores for AC sections from Group C MPC+HA joints wereless than corresponding HA sections but were not SS. In addition, whenthe ratios of the total Mankin scores for the HA injected andcontralateral HA+MPC injected joints from each group were calculated andplotted it was clearly evident that the 100 million dose of MPC was themost efficacious (FIG. 6).

The question of the sustainability of the 100 million MPC dose inpreserving joint cartilage integrity was addressed by studying themorphological, histological and biomechanical properties of the tissues22 and 50 weeks post injection ie, 24 and 52 weeks post meniscectomy. Asis evident from FIGS. 7 and 8 the difference between the mean values formorphological scores for HA and HA+100 million MPC diminish over thistime, although there is some evidence of a therapeutic effect at 24weeks. This view is supported by the HA/MPC+HA data which indicated astronger effect of the cells in suppressing osteophyte scores for up to52 weeks (FIGS. 10 and 11). On the other hand, similar plots for theMankin histopathology scores showed that by 52 weeks the protectiveeffects of the MPC was lost (FIG. 11).

Biomechanical indentation studies on the patella cartilages from jointsof all the animal groups were generally consistent with themorphological and histological data. However, the stiffness of thecartilage is influenced by the thickness of the cartilage which in theearly phases of injury may be hypertrophic but normalize with time. Thissituation may be occurring in the present model since the patellacartilage stiffness determined for the 25 and 100 million MPC groupswere significantly less than the 10 and 150 million MPC groups which,from other studies exhibited the most damage tissues (FIGS. 12 and 13).This interpretation was supported by the phase lag data which wassignificantly lower for the patellae from the 100 million MPC group bothrelative to the corresponding HA injected joints and the 150 million MPCdose (FIG. 14). Moreover, the mean phase lag values observed at 12 weekswere found to significantly increase at 24 and 52 weeks postmeniscectomy confirming the loss of a useful therapeutic effect of theinjected MPC beyond 6-12 months (FIG. 15). Phase lag reflects themolecular assembly of the cartilage extracellular matrix and the lowerthe angle (Phase) the greater the elasticity and thus ability to recoverfrom deformation (Cake et al., 2005).

The chondroprotective effects observed for the 100 mil MPC injectedjoints diminished with time; the positive effects noted at 12 and 24weeks BTM being lost by 52 weeks.

There was no evidence of synovial histopathology modulation. Clinicaland gross organ pathology conducted on these animals has not shown anyevidence of systemic adverse effect of MPC.

CONCLUSIONS

This is the first report, as far as we are aware, of a beneficialtherapeutic effect of allogeneic MPC on cartilage integrity in a modelof early OA. MPCs are known to release growth factors and cytokines andalso suppress the production of TNF-alpha by other cells, whileup-regulating anti-inflammatory cytokines (eg. IL-4, IL-10). Theseparacrine activities of MPC could stimulate chondrocyte biosynthesis ofnew matrix but also attenuate local production and activity of catabolicmediators. The finding in this study that 100 million MPC werechondroprotective was consistent with such a mechanism of action. Thedata generated in these sheep studies indicate that the duration of thechondroprotective effect mediated by a single intra-articular injectionof 100 million MPC is between 6-12 months post treatment suggesting thatmultiple injections may be required for the long term management of theOA patient.

While intra-articular injections of HA are widely used for the treatmentof knee osteoarthritis there is limited evidence that this therapy ischondroprotective (Ghosh et al., 2002). However, intra-articular HAtherapy is reported to provide symptomatic relief in OA which is of slowonset, but more sustained than with intra-articular corticosteroids(Bellamy et al., 2006).

Example 3 Relative Therapeutic Effects of Intra-Articular Injection ofHyaluronan (HA) or 100 Million Mesenchymal Precursor Cells (MPC)+HA onCartilage Integrity in a Model of Severe Osteoarthritis Induced byBilateral Total Medial Meniscectomy in Stifle Joints of OvariectomizedEwes

The knee joint meniscus performs an important role in protectingarticular cartilage (AC) against damage during normal joint articulation(Arnoczky et al., 1988). Total or partial excision of the meniscus inhumans following its injury generally results in premature degenerationof AC and progression to osteoarthritis (OA) (Jorgensen et al., 1987;Roos et al., 1998 and McNicholas et al., 2000). Experimental studieshave shown that unilateral or bilateral total meniscetomy in sheep alsoleads to premature breakdown of AC and the early onset of OA (Ghosh etal, 1990; Appleyard et al., 1999 and Ghosh et al., 1993c).

Since the failure of AC in meniscectomised joints is a consequence ofthe imposition of high focal and shearing stress on cartilage, bilateralmeniscectomy was found to induce a more rapid progression of cartilagedegeneration than unilateral meniscectomy where supportive pain-freeweight bearing can be accommodated by use of the contralateralnon-operated hind limb (Ghosh et al., 1993a and 1993b; Appleyard et al.,2003; Little et al., 1997 and Oakley et al., 2004). Furthermore,ovariectomised ewes subjected to bilateral meniscectomy have also beenshown to undergo a more progressive OA than adult castrated males(wethers), largely due to the depletion from their circulation of thecartilage protective hormone, oestrogen (Parker et al., 2003). For thesereasons ovariectomised and bilaterally meniscectomised ewes are favouredas a large animal model of OA to study the disease modifying activitiesof anti-OA agents (Ghosh et al., 1993; Smith et al., 1997; Burkhardt etal., 2001; Hwa et al., 2001 and Cake et al., 2000). Theovariectomised/bilaterally meniscectomised sheep model of OA wastherefore selected for the present investigation—the purpose of whichwas to evaluate the effects of intra-articularly (IA) administeredallogeneic Mesenchymal Precursor Cells (MPC) on induction of growth orregeneration of proteoglycan-rich cartilage and on chondroprotectionrelative to a currently used anti-OA therapy, IA Hyaluronan (HA).

Methods

Bilateral total medial meniscectomy (BTM) was undertaken in 18 adultMerino ewes that had undergone ovariectomy 3 months previously using apublished method (Cake et al., 1004). The surgical procedure andpost-operative regimen used for BTM was identical to that described forthe castrated male sheep BTM studies described in Example 2.

Twelve weeks post BTM, 6 ewes were sacrificed while the stifle (knee)joints of the remaining 12 meniscectomised ewes were randomly injectedwith either 2 mL high MW HA or 100 million MPC suspended in 2 mLProfreeze® plus 2 mL HA. This dose of MPC+HA was shown in Example 2 toafford the most beneficial chondroprotective effects in the BTM malesheep model. The meniscectomised and injected ewes were divided into twogroups of 6 that were sacrificed 24 and 36 weeks post-BTM, i.e. 12 and24 weeks post HA or MPC+HA intra-articular injection. To determine theeffects of gender on the response of AC joint destabilization 6untreated castrated male sheep were also subjected to BTM and sacrificed12 weeks post-meniscectomy.

At necropsy, joints were opened, menisci removed and the medial femoraland tibial plateux photographed. The recorded images were scored by 2blinded observers for gross morphological changes to cartilage using a0-4 scale. Synovium from the suprapatellar fold and a 5 mm wide coronalosteochondral slice were removed from the mid-line of the femur andtibia of each joint and processed for preparation of histologicalsections. Cartilage histopathology was assessed by two blinded observersusing a modified Mankin Scoring system as described previously (Littleet al., 1997). Synovial histopathology was scored using the criteriarecently described by Cake et al., 2008.

Histological serial sections from the same cartilage blocks as used forMankin Scoring were also utilized for histomorphometric analysis asdescribed previously (Caket et al., 2000; Cake et al., 2004). Thistechnique employs computer-aided image analysis (ImagePro Plus v.3.0,Media Cybernetics) to generate quantitative data on the dimensions andan index of the proteoglycan content of Toluidine blue stained AC. Inbrief, images of the stained sections were acquired via a MicrotekSlidescanner 35t plus (Microtek Model No. PTS-1950) at a resolution of1300 dpi and then analysed using Image J® software(http://rsb.info.nih.gov/ij/) on a personal computer. The digital imagesof the femoral and tibial sections were subdivided into inner, middleand outer regions, each region representing approximately one third ofthe total area of the cartilage sections. Spatial calibration of thesystem was achieved by scanning a 10×10 mm high precision reticule. Thisscale was then used to quantify the length (mm) and area (mm²) in ofeach region of the imported images. The average thickness of thesections was determined by dividing the area by the length. The opticaldensity (OD) of the TB stained cartilage sections was obtained as themean grey value (MGV) (sum grey values/number of pixels) and was takenas an index of proteoglycan (PG) content. The integrated grey-scaledensity (IGD) was calculated as MGV×regional area of section. Althoughthe grey scale system used was not independently calibrated against TBstained sections of known PG content, all histological sections were cuton the same microtome, were the same thickness and were processed as agroup using the same staining protocol. Differences in cartilagestaining are therefore relative rather than absolute. Intact patellaefrom all joints were removed within 1 hour of sacrifice and immediatelyfrozen and stored prior to topographical biomechanical indentationstudies to determine the stiffness and phase lag of the articularcartilage (Appleyard et al., 2003).

Statistical analysis to identify differences in treatments outcomes (HAversus HA+MPC) or treatments versus untreated 12 week post BTM controls,as assessed by the morphological and histological scoring systems, wasundertaken using Kruskal-Wallis nonparametric analysis and fordifferences between group comparisons using Maim Whitney U nonparametricanalysis with p<0.05 considered significant.

Data generated by the histomorphometric analysis of digitisedhistological sections were evaluated using the equal variance Two TailedStudent's T-Test with p<0.05 considered to be significant. Statisticalanalysis of patella cartilages biomechanical parameters with respect todifferent treatments and between time post-BTM was calculated using anindependent T-Test with p<0.05 considered significant.

Results

The gross morphological assessment of cartilage erosions and osteophyteformation in joints from the untreated ewes 12 weeks post-BTM confirmedthat this model of OA represented a more aggressive and severe form ofthe disease compared with meniscectomised castrated males subjected tothe same surgical procedure. For this untreated female control group themean cartilage morphological score for the femur was 87% and for thetibia 75% of the maximum scores used to assess this parameter. The grossmorphology scores obtained for the joints derived from the untreated 12week post meniscectomised castrated males subjected to the same surgicalprocedure was significantly less than for the ovariectomised ewes (FIGS.16 and 17) a finding which was consistent with previous observationsusing bilateral lateral meniscectomy (Parker et al., 2003; Cake et al.,2004).

While both treatments resulted in lower mean femoral morphologicalcartilage scores at 24 and 36 weeks than the baseline untreated 12 weekpost-meniscectomised ewes (data not shown), no significant differenceswere detected between the MPC and HA treated joints. We interpret thisto mean that in this model of severe OA, the severity of the grossmorphologic lesions (erosion and osteophyte scores) make theseparameters too insensitive to detect therapeutic differences.

Modified Mankin histopathology scores for cartilages from the untreated12 weeks post-BTM group were found to be consistent with the extent ofcartilage damage as assessed morphologically (FIGS. 16 and 17). Incontrast to morphologic parameters, at 36 weeks the total mean modifiedMankin score for the femoral cartilages in the ovariectomised ewes whoreceived MPC+HA was lower than the corresponding score for the jointsthat received HA alone and showed a significantly lower cell number(p=0.01) and a trend (p=0.06) for stronger inter-territorial ToluidineBlue (IT TB) staining for proteoglycans than HA alone (FIG. 18). Theseeffects were less pronounced for the tibial cartilages (FIG. 18).

The lower Modified Mankin histopathology cartilage score observed forthe MPC+HA injections at 36 weeks post meniscectomy relative to the HAinjected joints was highlighted when the ratio of the mean totalModified Mankin scores for the two intra-articular treatments weredetermined (FIG. 19). As each ratio was obtained from the two treatedjoints of the same animal a ratio=1 would indicate that both treatmentswere equally effective. However, for the ratios>1 the MPC+HA treatmentcan be said to be more beneficial. As is evident from FIG. 19 the meanof the ratios obtained for the femoral cartilages were significantlyhigher (1.71) than unity at 36 weeks post-BTM while the tibial cartilageratios (1.12) for the two treatments were only slightly in favor of theMPC+HA injected group (FIG. 19).

Next we examined the effect of the treatments on Mankin scores overtime. Significant differences in effects on femoral cartilage over timewere found between the MPC+HA and the HA alone treatment arms at 24 and36 weeks post meniscectomy, i.e. 12 and 24 weeks post-injection (FIG.20). In the group receiving MPC+HA, mean scores at 24 and 36 weeks wereprogressively lower than at the 12 week baseline. This was due toreduced scores and improvement in cell cloning (P=0.01) at 24 weeks, andin cell numbers (P=0.04) and inter-territorial Toluidine Blue stainingfor proteoglycans (PGs) (P=0.04) at 36 weeks relative to the 12 weekuntreated group (FIG. 20). No such improvements were seen in the HAalone group. No significant differences were observed between thesynovial pathology scores for any of the groups or intra-articulartreatments.

The analysis of cartilage thickness, area and intensity of TB stainingas an index of PG content for the 3 regions (inner, middle and outer) ofthe femoral condyles from the injected joints at 36 week post-BTM usinghistomorphometric methods of analysis are shown in FIG. 21. By 36 weekspost-BTM significant differences between treatments groups were evident.Femoral cartilages from the MPC+HA injected joints were significantlythicker (FIG. 21A) and occupied a significantly larger area (FIG. 21B)than the corresponding cartilages of HA injected joints. The largervolume of the femoral cartilages from the MPC+HA injected joints wasaccompanied by a higher content of proteoglycans as determined from theintegrated grey-scale density of the TB stained sections (FIG. 21C).

Again comparing these parameters in a time-based analysis, significantdifferences in effects on femoral cartilage over time were found betweenthe MPC+HA and the HA alone treatment arms at 24 and 36 weeks postmeniscectomy, i.e. 12 and 24 weeks post-injection. Using the samehistomorphometric methodology, we were able to demonstrate that theMPC+HA injection administered 12 weeks post meniscectomy resulted inprogressively greater proteoglycan-rich femoral cartilage growth orregeneration 12 and 24 weeks later (i.e. at 24 and 36 weeks post-BTM)than HA alone (FIGS. 22 to 24). Thus, femoral cartilages at 24 and 36weeks post-BTM from joints of meniscectomised ewes injected with MPC+HAat 12 weeks were significantly thicker (FIG. 22) and generally hadlarger areas (FIG. 23) than the baseline values from untreated joints at12 weeks post-meniscectomy. The corresponding regions scanned fromsections of femoral cartilage derived from HA injected joints failed todemonstrate statistically significant changes relative to the 12 weekuntreated controls (FIGS. 22 & 23). The integrated grey-scale density asa measure of PG content of sections of femoral cartilages wassignificantly higher for both HA and MPC+HA injected joints relative tothe same cartilage regions of joints from the untreated 12 week post-BTMgroup but the magnitude of the MPC+HA induced change was significantlygreater than HA alone (FIG. 24). Whereas the MPC+HA group developedalmost 60% greater proteoglycan-rich femoral tissue at 36 weeks comparedwith baseline (P<0.001), and this rate of cartilage growth had notreached a plateau phase, the HA only group had reached a plateau phaseand developed only about 30% greater tissue. This indicated thattreatment with MPC+HA stimulated significantly greater increase inproteoglycan-rich cartilage over the 24 week period of follow-up (i.e.growth and/or regeneration of cartilage) relative to both baseline andto any temporal effects of HA treatment alone.

The results of the indentation studies on the patella cartilages fromthe injected joints failed to demonstrate any difference in thebiomechanical properties of the cartilages for the two treatments butchanges were identified with respect to time elapsed post meniscectomyand the untreated 12 week post-BTM group. The stiffness of the patellacartilages from the MPC+HA at 24 weeks post-meniscectomy wassignificantly higher than at 12 weeks (P=0.05) and 36 weeks (P<0.01)(data not shown). However, both treatments produced thicker patellacartilage 36 weeks compared to 24 weeks (P=0.001) that was also lowerthan the non-treated 12 week control (P=0.01). Patella cartilagephase-lag for both treatment groups at 24 and 36 weeks were higher thanthe untreated 12 week controls (P=0.001) (data not shown).

Discussion

The present studies have shown that bilateral medial meniscectomy inovariectomised ewes induced pathological changes in joint articularcartilage after 3 months that were consistent with progressive andsevere OA. Thus the gross morphology scores for the femoral and tibialcartilages were 87-70% of the maximum score. Interestingly, castratedmales subjected to the same surgical procedure and sacrificed at thesame time (12 weeks) showed less severe cartilage lesions than theobserved for the ovariectomised females. The extent of cartilagepathology was also reflected in the high aggregate Modified Mankinhistopathology scores observed for this group that were consistent withthe assignment of early OA (Little et al., 1997). Although previousstudies had identified a strong association of OA in postmenopausalfemales, which was explained by the depletion of estrogen from thecirculation (Roos et al., 2001; Pelletier et al., 2007 and Nevitt etal., 1996) and was supported by studies in ovariectomised ewes (Parkeret al., 2003 and Cake et al., 2004), other more recent studies suggeststhat the adipose derived hormone, Leptin, may play a more significantrole in mediating cartilage breakdown and OA (Dumond et al, 2003 andTeichtahl et al., 2005). The 3 months post BTM period was thereforetaken as the starting point for the evaluation of the relative effectsof intra-articular injections of HA or MPC+HA on the rate of progressionof cartilage pathology 12 and 24 weeks following the administration ofthese agents.

The results of this study indicated that a single intra-articularinjection of 100 million MPC dispersed in 2 mL HA and 2 mL Profreeze® (acommercial cryoprotectant) into joints with established, severe OA can,over an intervening period of 24 weeks, slow the progression of jointpathology and enhance growth and/or regeneration of proteoglycan-richcartilage to a greater extent than a single injection of 2 mL HA.Surprisingly, the growth/regenerative and chondroprotective effectsmediated by the MPC were observed to be more significant 24 weeks afteradministration than after 12 weeks in the majority of parametersexamined, indicating progressive effects which had not yet reached aplateau phase. The reasons for this finding are presently unclearhowever, it is possible that the growth factors such as members of theTGF-beta superfamily, eg BMPs, released by the MPC (Ahrens et al., 1993;Aggarwal et al., 2005) were supportive of the anabolic (compensatory)phase of cartilage to the altered mechanical stresses imposed across thejoint by medial meniscectomy. This view was supported by thehistomorphometric data that demonstrated the presence of higher volumesand more intense staining for proteoglycans in the MPC injected groupsthan at the commencement of treatment at 12 weeks post-BTM. These matrixchanges are consistent with increase chondrocyte biosynthesis.Significantly, the magnitude of the anabolic parameters was generallyfound to be greater in the cartilages of animals who received the MPC+HArather than HA alone. The ability of MPC to preserve and even enhancethis cartilage response to mechanical overload contrasts with the knowninhibitory effects on chondrocyte metabolism mediated by manytraditional treatments of OA, including many of the steroidal andnon-steroidal anti-inflammatory drugs (NSAIDs) (McKenzie et al., 1976;Ghosh, 1988; Brandt, 1993 and 1993a; Huskisson et al., 1995).

Multiple intra-articular HA injections have been used as a therapy forthe management of knee OA for more than 30 years. Although the consensusis that this form of treatment does provide symptomatic reliefclinically, a recent review and a meta-analysis of published HA clinicaltrials have questioned the validity of this conclusion on the basis ofthe stronger placebo effects associated with intra-articular injections,difficulty of blinding investigators and publication biases (Brandt etal., 2000; Lo et al., 2003). Whether intra-articular HA exhibits anychondroprotective or cartilage regenerative activity is alsocontroversial. However, extensive animal investigations have shown thatHA does exhibit analgesic, anti-inflammatory and disease modifyingeffects in rabbit and ovine models of OA induced by uni-lateral andbilateral meniscectomy as well as anterior cruciate ligament transectionin dogs. A discussion of these data together with preclinical andlaboratory based clinical studies with HAs of different molecular weighthas been reviewed (Ghosh et al., 2002).

In the present study only a single intra-articular injection of HA,either alone or in combination with MPC, was evaluated. On the basis ofour own data we conclude that the long-lasting growth and regenerative,as well as chondroprotective, effects afforded by the MPC+HA combinationwas mediated by the MPC. In this regard it is important to note that thedesign of this study allowed each animal to act as its own control sinceone joint received HA while the contra-lateral joint received the samequantity of HA plus the MPC in the cryoprotectant, Profreeze®. Sinceboth knee joints were surgically de-stabilised in the present study andwere injected at the same time we are confident that the magnitude andnature of the weight-bearing mechanical stresses acting on the articularcartilages was the same on both joints.

From the present studies we conclude that a single intra-articularadministration of MPC+HA into ovine joints with pre-existing, severe OAresults in growth or regeneration of proteoglycan-rich cartilage asmanifest by increased cartilage extracellular matrix 24 weeks posttreatment relative to baseline pre-treatment and to HA injectedcontrols.

Example 4 Ovine Disc Re-Generation Studies Using Immunoselected MPCMethods

Thirty-six age-matched, Merino wethers (approximately 18 to 24 monthsold) were used for this study. In all 36 sheep three adjacent lumbardiscs (L3-L4, L4-L5, L5-L6) were injected with 1.0 IU chondroitinase ABC(Seikagaku Corporation, Japan) in approximately 0.1 ml sterile normalsaline to breakdown and remove the PGs of the NP. The remaining lumbardiscs (L1-L2 and L2-L3) were not injected with chondroitinase ABC andserved as controls. Fifteen weeks (±3 weeks) following administration ofchondroitinase ABC, injections MPCs (0.5×10⁶ cells) in ProFreeze™Freezing Medium (NAO) or ProFreeze™ NAO alone (Lonza Walkersville Ltd.)mixed with an equal volume of hyaluronic acid (Euflexxa®, (FerringPharmaceuticals) were administered directly into the chondroitinase ABCtreated nuclei pulposi of the intervertebral discs identifiedschematically in FIG. 25. The respective experimental groups weresacrificed 3 and 6 months later as summarized in Table 1.

TABLE 1 Study Design Summary 15 ± 3 weeks Baseline Group No. Disc beforeBaseline Day 0 Sacrifice Analysis at Sacrifice 1 n = 6 L1-L2 Noinjection No injection 3 mths Compositional/Histology L2-L3 No injectionNo injection 3 mths Compositional/Histology L3-L4 Chondroitinase MPCs0.5 × 10⁶ 3 mths Compositional/Histology L4-L5 Chondroitinase Noinjection 3 mths Compositional/Histology L5-L6 Chondroitinase HA and NAO3 mths Compositional/Histology 2 n = 6 L1-L2 No injection No injection 6mths Compositional/Histology L2-L3 No injection No injection 6 mthsCompositional/Histology L3-L4 Chondroitinase MPCs 0.5 × 10⁶ 6 mthsCompositional/Histology L4-L5 Chondroitinase No injection 6 mthsCompositional/Histology L5-L6 Chondroitinase HA and NAO 6 mthsCompositional/Histology

Animals had lateral plain radiographs taken of the lumbar spine underinduction anaesthesia at the following time points: Day 0 (Injection ofchondroitinase ABC (Seikagaku Corporation, Japan), Day of Test Articleadministration (15±3 weeks following induction of lumbar discdegeneration) and 3 months and 6 months following implantation of theTest Article. Evaluation of the radiographs was undertaken using anindex of intervertebral height (DHI) calculated by averaging themeasurements from the anterior, middle and posterior parts of the IVDand dividing it by the average of the adjacent intervertebral bodyheights as described by (Masuda et al., 2004).

The MRIs were taken of the lumbar spine under induction anaesthesia atthe following time points: Day Zero (injection of chondroitase ABC[Seikagu Corp Japan]), Day of test article administration (15+3 weeksfollowing induction of lumbar disc degeneration), 3 months and 6 monthsfollowing implantation of test article. Disc were graded from the MRIscans using the Pfirrmann Classification System (Pfirrmann et al.,2001).

Spinal motion segments that were designated for histochemical andbiochemical analysis were isolated by cutting through the cranial andcaudal vertebral bodies close to the cartilaginous endplates using abone saw. These spinal sections were fixed en bloc in Histochoice® for56 h and decalcified in several changes of 10% formic acid in 5% NeutralBuffered Formalin for 2 weeks with constant agitation until completedecalcification was confirmed using a Faxitron HP43855A X-ray cabinet(Hewlett Packard, McMinnville, USA).

Multiple sagittal slices of the decalcified specimens, approximately 5mm thick, were dehydrated through graded ethanol solutions by standardhistological methods and embedded in paraffin wax. Paraffin sections 4μm thick were mounted on Superfrost Plus glass microscope slides(Menzel-Glaser), dried at 85° C. for 30 min then at 55° C. overnight.The sections were then deparaffinised in xylene (4 changes×2 min) andrehydrated through graded ethanol washes (100-70% v/v) to tap water. Onesection from all blocks prepared from the sagittal slices was stainedwith haematoxylin and eosin. The coded section was examined by anindependent histopathologist who compared the histologicalcharacteristics of those levels that were subjected to enzyme injectiononly with those that were enzyme-injected and subsequently receivedMPCs. A four-point semi-quantitative grading system was used to assessthe microscopic features of the entire disc as shown in Table 2.Additional tinctorial stains including Alcian Blue (for generalglycosaminoglycan species) and Safranin 0 (specific for chondroitinsulphate species) were also prepared to demonstrate the extent of discmatrix synthesis.

The immunohistochemistry procedures were also performed using a Sequenzacassette and disposable Coverplate immunostaining system as describedpreviously (Melrose et al., 2003; Melrose et al., 2002; Melrose et al.,2000; Melrose et al., 2002a; Melrose et al., 1998; Panjabi et al., 1985;Race et al., 2000; Smit, 2002). Endogenous peroxidase activity wasinitially blocked by incubating the tissue sections with 3% H₂O₂. Theywere then pre-digested with combinations of chondroitinase ABC (0.25U/ml) in 20 mM Tris-acetate buffer pH 8.0 for 1 h at 37° C., bovinetesticular hyaluronidase 1000 U/ml for 1 h at 37° C. in phosphate bufferpH 5.0, followed by three washes in 20 mM Tris-HCl pH 7.2 0.5M NaCl(TBS) or proteinase-K (DAKO S3020) for 6 min at room temperature toexpose antigenic epitopes. The tissues were then blocked for 1 h in 20%normal swine serum and probed with a number of primary antibodies tolarge and small proteoglycans and collagens (Table 3). Negative controlsections were also processed either omitting primary antibody orsubstituting an irrelevant isotype matched primary antibody for theauthentic primary antibody of interest. Commercial (DAKO) isotypematched mouse IgG (DAKO Code X931) or IgM (DAKO Code X942) controlantibodies (as appropriate) were used for this step. The DAKO productsX931 and X942 are mouse monoclonal IgG₁ (clone DAK-GO1) and monoclonalIgM (clone DAK-G08) antibodies directed against Aspergillus nigerglucose oxidase, an enzyme that is neither present nor inducible inmammalian tissues. Horseradish peroxidase or alkaline phosphataseconjugated secondary antibodies were used for the detection using 0.05%3,3′-diaminobenzidene dihydrochloride and 0.03% H₂O₂ in TBS, Nova RED,nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate/iodonitrotetrazolium violet (NBT/BCIP/INT) or New Fuchsin as substrates. Thestained slides were examined by bright field microscopy and photographedusing a Leica MPS 60 photomicroscope digital camera system.

TABLE 2 Grading system of histologic changes in lower lumbar discs (BEPbony end-plate, CEP cartilaginous end-plate) Grade Annulus fibrosisNucleus pulposus Cartilage end-plate Margins/subchondral bone 1 Intactlamellae Homogeneity Uniform thickness Even thickness of BEP Narrowinter-lamellar matrix Absence of clefting Intact attachment to boneLamellar bone only Intact annulus attachment Uniform calcification <⅕Distinct junction with CEP Vessels only in outer ⅓ of depth Few vascularintrusions into Uniform cell distribution CEP 2 Minor lamellar splittingand Minor clefting Minor cartilage thinning Slightly uneven BEPdisorganisation. Minor widening of Minor cell necrosis Small transversefissures Schmorl's nodes matrix Minor disorganisation of Minor posteriordisplace- Irregular thickening of calcified Minimal remodelling of BEPattachment Rim lesion without ment of annulus zone Small marginalosteophytes reparative reaction Minor chondrone formation Few invadingvascular channels Small chondrones 3 Moderate widening of matrixModerate clefting Marked cartilage thinning Moderately uneven BEPmoderate fissuring of attachment Moderate cell necrosis Markedthickening of calcified Vascularised Schmorl's nodes Radiating tears notinvolving outer Cystic degeneration zone Moderate trabecular thickening⅓ minimal chondroid metaplasia Posterior displacement within Manytransverse fissures Defect in bone lamellae Cystic degeneration Vesselsin annulus Many vascular channels Minimal fibrosis tissue in outewr andmiddle ⅓ rim lesion Centripetal extension of Many chondrones marrowspaces with minor reparative reaction collagen Medium-size osteophytesModerate chondrone formation 4 Extensive lamellar disorganisationComplete loss of nucleus Total loss of cartilage Marked uneven BEPRadiating tears extending into outer Loose body formation Calcificationof residual Ossified Schmorl's nodes ⅓ Marked chondrone formationcartilage Large osteophytes Extensive chondroid metaplasia Widespreadfissuring Marked trabecular thickening Vessels in all zones Markedfibrosis of marrow spaces Rim lesion with marked reparative Cartilageformation reaction

TABLE 3 Primary antibodies to proteoglycan and collagen core proteinepitopes Primary antibody epitope Clone (isotype) References Large PGsAggrecan AD 11-2A9 (IgG) 26, 30 Versican 12C5 (IgG) 26, 28 Collagen TypeI I8H5 (IgG₁) 23, 28 Type II II-4CII (IgG₁) 28 Type IV CIV-22 (IgG₁) 28Type VI Rabbit polyclonal 28 Type IX Mouse monoclonals 35 D1-9 (IgG₁),B3-1 (IgG_(2b))

Samples of annulus fibrosus and nucleus pulposus were dissected from theprocessed blocks finely diced and representative portions of the tissuezone of known wet weight were freeze dried to constant weight.Triplicate portions (1-2 mg) of the dried tissues were hydrolysed in 6MHCl at 110° C. for 16 h and aliquots of the neutralised digests assayedfor hydroxyproline as a measure of the tissue collagen content (Sakai etal., 2005). Triplicate portions of dried tissues (˜2 mg) will also bedigested with papain and aliquots of the solubilised tissue assayed forsulphated glycosaminoglycan using the metachromatic dye1,9-dimethylmethylene blue as a measure of PGs (Sakai et al., 2005).

The motion segments were wrapped in saline-soaked gauze, sealed indouble thickness polythene bags and frozen at −30° C. untilbiomechanical testing. This treatment has been shown not to alter thebiomechanical characteristics of the tissue (Panjabi et al., 1985).Biomechanical testing was undertaken to measure the stiffness of eachdisc in axial compression, flexion, extension, lateral bending and axialtorsion under defined computer-controlled conditions approximatingphysiological loading (Panjabi et al., 1985; Race et al., 2000; Smit,2002; Wilke et al., 1999). Full details of the testing protocol aredocumented elsewhere (Panjabi et al., 1985; Race et al., 2000; Smit,2002; Wilke et al., 1999). The specimens for testing (functional spinalunits, FSUs) comprised two adjacent vertebrae, the intervening disc andassociated ligaments. Three FSUs per spine were tested: a level that wasonly degraded with C-ABC only, one in which the disc was degraded withC-ABC and which was subsequently treated with hyaluronic acid only andthe central level that was degraded with C-ABC and which wassubsequently treated with hyaluronic acid and with MPCs. Each FSU wasmounted in two aluminium alloy cups and secured with three bolts andcold cure polymethyl methacrylate dental cement (Vertex SC Self Curing,Dentimex BV, Zeist, Holland). Care was taken to ensure that the midlineof the intervertebral disc is positioned horizontally. The motionsegments will be centred in the cups by placing a dowel through thevertebral canal into a hole in one of the cups. All tests were conductedin a saline water bath maintained at 37° C. Prior to the commencement oftesting each FSU will be preloaded to a stress of 0.5 MPa until areproducible state of hydration is achieved. This was used as thebaseline prior to each test. The preload stress of 0.5 MPa simulatesrelaxed standing and was based on in vivo measurement of intradiscalpressure.

Mechanical tests were performed using a Model 8511 DynamicServohydraulic Materials Testing Machine (INSTRON Pty Ltd, High Wycombe,UK) equipped with a ‘six degrees of freedom’ load cell to allow thesimultaneous monitoring and control of forces in all three planes. Themachine was controlled by a personal computer and custom-designedsoftware that also records and analyses the data. Test data was acquiredin stable hysteresis from the final of five sinusoidal 0.1 Hz loadingcycles in either axial load or torsion control. The tests were performedare pure axial compression, left and right lateral bending, combinedflexion/extension and pure axial torsion.

Pure axial compression to 200N was produced in the FSU with little or nobending or flexion accompanying the load. All compressive tests wereperformed using point contact on the cranial cup surface. The neutralaxis of bending (NAB) is determined by applying a cyclic load to thejoint through a point on the aluminium alloy cup holding the specimen toachieve negligible bending. This trial and error process enables asclose to pure axial compression as possible using a rigid point loadcontact. Despite slight variability between specimens this point isfound on the sagittal plane approximately 10 mm anterior to the spinalcanal but slightly posterior to the disc centroid. Marks were placed 10mm anterior and posterior and to the left and right of the NAB toposition the offset loads for the bending tests. A maximum compressiveload of 200 N was applied at each point to produce 2 Nm of bending and200 N of axial compression.

Conservative bending and compressive loads were chosen to ensure thatthe disc, posterior elements, endplates and other ligamentous structureswere not e damaged. Pure bending was not produced using this loadingmethod. Instead a combination of bending and axial compression waspresent for the combined flexion/extension and lateral bending tests. Webelieve this was justified given that in vivo loading would seldomproduce pure bending but rather a combination of compression andbending. In either load case, all loads were applied consistently toeach specimen allowing direct comparisons of the mechanical response.

For the torsion tests 5 Nm of pure axial torsion will be applied. Thiswas within the physiological range of torques estimated from, andapplied in, other studies. A novel custom designed torsion testingsystem will be used to apply pure torsion to each FSU. This system usesa ballscrew/thrust plate mechanism to convert the axial displacement ofthe Instron actuator into pure rotation. An X-Y bearing table ensuresthat the FSU does not have a fixed centre of rotation imposed on itduring testing. This is important, as the centre of rotation is notconstant during axial rotation. The inferior cup was fixed to a torquetransducer with the superior cup fixed to the X-Y bearing table andballscrew/thrust plate mechanism.

All tests were conducted on the intact FSU initially. Once completed thedisc were isolated by cutting through the posterior elements using asmall hacksaw blade passed through the neural foramen and cuttingposteriorly. This cut through the zygapophysial joints and theinterspinous and supraspinous ligaments, leaving the intervertebraldisc, the posterior and the anterior longitudinal ligaments intact. Thecut was made in a wedge fashion increasing posteriorly to ensure nocontact between the zygapophyseal joints. All tests were then repeatedon the isolated disc.

Data analysis included parameters such as stiffness in the linear regionduring the fifth loading cycle, hysteresis and strain energy and theextent of the neutral zone. Data from the control levels was comparedwith the degenerated/MPC-injected levels and repeated measures analysisof variance was conducted on each of the biomechanical parameters.

Results

All animals in the MPC injected groups maintained normal body weightsand showed no evidence of adverse side effects over the duration of theexperiment.

In the Chondroitinase-ABC injected discs the depletion of PGs by thisenzyme resulted in a 38% decrease in disc height index (DHI) in allinjected discs after 3 months. This loss of disc height confirmed thedegenerate status of the nucleus pulposus prior to treatments andhitherto is referred to as the pre-MPC DHI. Three months post HA orMPC+HA injection into the degenerate discs failed to produce anysignificant increase in DHI relative to the pre-MPC DHI (FIG. 26).However, by 6 months post treatment, discs injected with MPC+HA showed amean increase of 52% in DHI relative to the corresponding 3 month scores(Group 1) (FIG. 26 and Table 4). In contrast, discs injected with HAalone only showed a 23.1% mean improvement in the DHI scores over thesame period (FIG. 26 and Table 4). Significantly, the mean DHI of thelow MPC+HA injected discs were comparable 6 months post treatment to theDHA scores for the non-chondroitinase ABC injected (ie, non-degenerate)control discs (FIG. 26).

A statistical analysis for the DHI for 6 versus 3 months post HA orMPC+HA injection is shown in Table 4.

Administration of ovine MPC together with a suitable carrier, such ashigh molecular weight hyaluronic acid (HA), into the nucleus pulposus ofexperimentally created degenerate IVDs has been shown in the presentexperiments to accelerate the regeneration of the disc extracellularmatrix as assessed radiographically by the recovery of disc height. Thisinterpretation is based on the assumption that in the loaded spinalcolumn the disc height is maintained by the presence within the NP andinner-annulus of high concentrations of matrix proteoglycans thattogether with their bound water molecules confer a high swellingpressure to this structure. Indeed, the use of chondroitinase-ABC toinduce disc degeneration at the commencement of these experiments reliedon the ability of this enzyme to degrade and remove the majority of theproteoglycans from the NP extracellular matrix.

The data obtained to date suggests that the therapeutic effect mediatedby the MPC is a relatively slow process. In the present study, the doseof 0.5×10⁶ MPC was particularly effective.

Although the present experiments were terminated 6 months after the MPCwere injected into the disc, the level of disc height recovery obtainedfor the low dose MPC injections was found to be close to the valuesobserved for the non-chondroitinase ABC injected internal controls,suggesting that the maximum extent of NP reconstitution was achievedover this period.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

All publications discussed and/or referenced herein are incorporatedherein in their entirety.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of this application.

TABLE 4 Extent of disc height restoration 3 and 6 months postintra-discal injection of Mesenchymal Precursor Cells (MPC) + Hyaluronan(HA) or HA alone into degenerate sheep lumber discs PRE-MPC 3 MONTHSPOST 6 MONTHS POST INJECTION DHI MPC INJECTION DHI MPC INJECTION DHINon- cABC + Non- cABC + Non- cABC + in- cABC cABC + HA + in- cABC cABC +HA + in- cABC cABC + HA + jected only HA MPC jected only HA MPC jectedonly HA MPC MEAN 0.054 0.04 0.04 0.04 0.055 0.0416667 0.04333330.0383333 0.0566667 0.0516667 0.0533333 0.05833333 Std Deviation 0.0150.01 0.01 0.01 0.01 0.014 0.008 0.014 0.015 0.017 0.008 0.007 % Changefrom 0.2 23.9 23.1 52.174 3 months to 6 months Statistical 0.83 0.310.059 0.0142 Significance (P values) P < 0.05 = significant DHI = DiscHeight Index cABC = Chondroitinase ABC

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1. A method of treating and/or preventing a disease in a subject arisingfrom degradation and/or inflammation of connective tissue, the methodcomprising administering to the subject MPCs and/or progeny cellsthereof and/or soluble factors derived therefrom.
 2. The method of claim1 wherein the connective tissue is rich in proteoglycans.
 3. The methodof claim 1 or claim 2 wherein the connective tissue is cartilage.
 4. Themethod of claim 3 wherein the disease results in one or more defects inthe cartilage.
 5. The method of claim 4 wherein the MPCs and/or progenycells and/or soluble factors are not directly administered into acartilage defect.
 6. The method of any one of claims 1 to 5 wherein theMPCs and/or progeny cells and/or soluble factors are administered to ajoint space.
 7. The method of claim 6 wherein the joint space is in aknee joint, hip joint, ankle joint, shoulder joint, elbow joint, wristjoint, hand or finger joint or a joint of the foot, or an invertebraldisc joint.
 8. The method of claim 6 or claim 7 wherein the MPCs and/orprogeny cells and/or soluble factors are administered by intra-articularinjection.
 9. The method of any one of claims 1 to 8 wherein theadministration of MPCs and/or progeny cells and/or soluble factorsresults in preservation or generation of cartilage that is rich inproteoglygasn.
 10. The method of claim 9 wherein the cartilage that isrich in proteoglycan is hyaline cartilage.
 11. The method of any one ofclaims 1 to 10 wherein the disease is tendonitis, back pain, rotary cufftendon degradation, Carpal tunnel syndrome, DeQuervain's syndrome,degenerative cervical and/or lumbar discs, intersection syndrome, reflexsympathetic dystrophy syndrome (RSDS), stenosing tenosynovitis,epicondylitis, tenosynovitis, thoracic outlet syndrome, ulnar nerveentrapment, radial tunnel syndrome, repetitive strain injury (RSI),osteoarthritis, rheumatoid arthritis, psoriatic arthritis, seronegativearthritis, arthritis associated with inflammatory bowel disease orankylosing spondylitis and degenerate invertebral disc disorders. 12.The method of any one of claims 1 to 11 which further comprisesadministering hyaluronic acid (HA).
 13. A composition comprising; i)supernatant, or one or more soluble factors, derived from mesenchymalprecursor cells (MPCs) and/or progeny cells thereof, and ii) hyaluronicacid.
 14. A composition comprising; i) mesenchymal precursor cells(MPCs) and/or progeny cells thereof, and ii) hyaluronic acid.
 15. Use ofsupernatant, or one or more soluble factors, derived from mesenchymalprecursor cells (MPCs) and/or progeny cells thereof for generating,repairing and/or maintaining connective tissue in a subject.